gs expression system bibliography in progress version...

GS Bibliography (March 2013) Revision 30 1 Lonza

BIBLIOGRAPHY

(REVISION 30) Lonza has compiled a list of scientific papers relating to the use of the GS Gene Expression System™. For your convenience, we have grouped these papers into the following categories: 1. SECTION 1: GS Expression Methodology and Cell Line Selection Strategies

2. SECTION 2: Cell Line Stability

3. SECTION 3: Antibody Production

4. SECTION 4: Recombinant Proteins (non-antibody) from NS0 Cells

5. SECTION 5: Recombinant Proteins (non-antibody) from CHO Cells

6. SECTION 6: Product Characteristics and Critical Quality Attributes

7. SECTION 7: Process Conditions for GS Cell Lines, including Media and

Feeding Strategies

8. SECTION 8: Metabolism of GS Cell Lines

9. SECTION 9: Virology of NS0 Cell Lines

10. SECTION 10: ‘Omic studies of GS Cell Lines and Systems Biology

11. SECTION 11: Cell Line Engineering

♦ Some references will be found in more than one category. ♦ The file number to the right hand side of the reference is for Lonza’s internal use

only. To help scan through the literature, there is a summary (if applicable) after each

reference which has been written by Lonza. The summary aims to provide a brief interpretation of the information within the publication. We do recommend, however, that you read the particular reference for yourself, in order to draw your own conclusions about the information provided in the publication of interest.


FILE

NUMBER

SECTION 1: GS Expression Methodology and Cell Line Selection Strategies

Astley K , Naciri M, Racher AJ, Al-Rubeai M. (2007) The role of p21cip1 in adaptation of CHO cells to suspension and protein-free culture. J Biotechnol 130:282-290

158

Astley K, Al-Rubeai M (2008) The role of Bcl-2 and its combined effect with p21CIP1 in adaptation of CHO cells to suspension and protein-free culture. Appl. Microbiol. Biotechnol. 78:391-399.

169.

Barnes LM, Bentley CM, Dickson AJ (2000) Advances in animal cell recombinant protein production: GS-NS0 expression system. Cytotechnol. 32:109-123. ♦ General review of the GS expression system including history of the NS0 cell

line.

100.

Barnes LM , Bentley CM, Moy N, Dickson AJ (2007) Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells. Biotechnol. Bioeng. 96:337-348. ♦ The paper describes a study, by Northern, Southern and copy number analysis,

of the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. This article discusses these findings in relation to vector design.

141.

Bebbington CR (1991) Expression of antibody genes in non-lymphoid mammalian cells. Methods in Enzymol. 2:136-145. ♦ Describes GS vector for use in CHO cells and antibody production in excess of

200 mg/L.

1.

Bebbington CR , Hentschel CCG (1987) The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells. DNA cloning, Glover D (ed.) Acad. Press N.Y. 3:163-180. ♦ Practical methods for use of GS expression systems in CHO.

2.

Bebbington CR , Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnol. 10:169-175. ♦ Describes use of GS selection system in NS0 for production of recombinant

antibody (cB72.3) to yields of 560 mg/L in fed batch airlift.

3.

Bebbington CR (1995) Expression of antibody genes in mammalian cells. In: "Monoclonal antibodies: the next generation" Zola H (ed.). Bios Scientific (Oxford) pp165-181. ♦ Review of expression systems including GS. Also discusses glycosylation of

antibodies.

71.


Bebbington CR (1995) Use of vectors based on gene amplification for the expression of cloned genes in mammalian cells. In: DNA cloning (3 ed.) Hames D and Glover D (2nd ed.) pp85-111. ♦ Methods descriptions including GS.

73.

Bloom JW, Madanat MS, Marriott D, Wong T, Chan SY. (1997) Intrachain disulfide bond in the core hinge region of human IgG4. Protein Sci. 6:407-415.

217.

Brand HN, Froud SJ, Metcalfe HK, Onadipe AO, Shaw A, Westlake AJ (1994) Selection strategies for highly productive recombinant cell lines. In: Animal Cell Technology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, Griffiths JB and Belthold W, Butterworth-Heinemann pp55-60. ♦ Compares productivity of attached and suspended GS-CHO. Reviews criteria

for selecting highly productive GS-NS0 and GS-CHO cell lines.

7.

Cockett MI, Bebbington CR, Yarranton GT (1991) The use of engineered EIA genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucl. Acids. Res. 19:319-325. ♦ TIMP expressed in GS-CHO with EIA. In the presence of EIA non amplified

lines gave levels comparable with gene amplified GS-CHO lines (110 µg/106

cells/24h). EIA also enhanced production of procollagenase (13 µg/106

cells/24h).

14.

Davies SL, O'Callaghan PM, McLeod J, Pybus LP, Sung YH, Rance J, Wilkinson SJ, Racher AJ, Young RJ, James DC (2011) Impact of gene vector design on the control of recombinant monoclonal antibody production by Chinese hamster ovary cells. Biotechnol. Prog. 27:1689-1699.

203.

Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC. (2013) Functional heterogeneity and heritability in CHO cell populations. Biotechnol. Bioeng. 110:260-274.

218.

de la Cruz Ed monds MC , Tellers M, Chan C, Salmon P, Robinson DK, Markusen JF. (2006) Development of transfection and high-producer screening protocols for the CHOK1SV cell system. Mol. Biotechnol. 34:179-190.

175.

Froud SJ (1994) Selection strategies for highly productive recombinant lines. ♦ Paper read at 'Commercializing human monoclonal antibodies' conference. San

Diego, Feb 7-8 1994. Describes strategies for isolating cell lines making up to 1300 mg of recombinant antibody/litre.

18.

Galbraith DJ, Tait AS, Racher AJ, Birch JR, James DC (2006) Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnol. Prog. 22:753-762. ♦ Describes optimisation of polyethyleneimine-mediated transfection as a method

for the very rapid production of milligramme quantities of antibody.

134.

Hayward BE, Hussain A, Wilson RH, Lyons A, Woodcock V, McIntosh B, Harris TJR (1986) The cloning and nucleotide sequence of cDNA for an amplified glutamine synthetase gene from the chinese hamster. Nucl. Acids. Res. 14:999-1008.

79.


Hovey A, Bebbington CR, Jenkins N (1994) Simultaneous control of growth and productivity using a mutant CHO cell line. In: Animal Cell Technology: Products of today, prospects for tomorrow. Eds: Spier RE, Griffiths JB, Berthold W. Butterworth – Heinemann, pp422-424. ♦ Heat shock induction used to produce recombinant TIMP from a mutant CHO

cell line. TIMP was expressed in a GS vector.

70.

Hovey A, Bebbington CR, Jenkins N (1994) Control of growth and recombinant protein synthesis by heat-shock in a mutant mammalian cell line. Biotechnol. Letters 16:215-220. ♦ Describes use of heat shock to induce recombinant protein (TIMP) synthesis in

a temperature sensitive CHO cell line. TIMP expressed in a GS vector.

75.

Jenkins N , Hovey A (1993) Generation of CHO cell mutants for growth control. In: Animal Cell Technology: Basic and Applied Aspects 5. Eds: Kaminogawa S et al pp267-272. ♦ Created temperature sensitive mutant of CHO expressing TIMP from GS vector.

Temperature switching arrested cell growth and increased product yield.

76.

Jenkins N , Hovey A (1993) Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotech. Bioeng. 42:1029-1036. ♦ Temperature sensitive mutants of CHO cells were transfected with the gene for

TIMP using GS amplification. Alternating incubation between permissive (34oC)

and non permissive (39oC) temperatures arrested cell growth whilst maintaining high cell viability for up to 170 hours. TIMP production rate was increased 3 to 4

fold over the period of arrest compared with controls at 34oC resulting in a 35% increase in yield.

69.

Kalwy S, Rance J, Young R (2006) Toward more efficient protein expression: keep the message simple. Mol. Biotechnol. 34:151-156. ♦ Discusses benefits of optimising gene coding sequence, eg by removal of

cryptic splice sites or mRNA-destabilising motifs, for improving productivity.

140.

Kennard ML, Goosney DL, Monteith D, Roe S, Fischer D, Mott JE (2009) Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology. Biotechnol. Bioeng. 104:526-539. ♦ Describes head-to-head comparison of CHOK1SV with CHO DG44 and CHO-S

as hosts for antibody expression using an artificial chromosome vector. Antibody concentrations in excess of 1.2 g/L for CHOK1SV-derived cell lines, whereas concentrations for the CHO-S and DG44 cell lines were in the range 0.1 to 0.48 g/L.

180.

Kennard ML , Goosney DL, Monteith D, Zhang L, Moffat M, Fischer D, Mott JE (2009) The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technology. Biotechnol. Bioeng. 104:540-553.

181.

Kingston RE, Kaufman RJ, Bebbington CR, Rolfe MR (1992) Amplification using 33.


CHO cell expression vectors. In: Current Protocols in Molecular Biology Supplement 18 Unit 16.14. ♦ Protocol for use of GS (and other expression vectors) in CHO cells.

Lindgren K , Salmén A, Lundgren M, Bylund L, Ebler Å, Fäldt E, Sörvik L, Fenge C, Skoging-Nyberg U. (2009) Automation of cell line development. Cytotechnology 59:1-10.

179.

Marchant RJ, Al-Fageeh MB, Underhill MF, Racher AJ, Smales CM (2008) Metabolic rates, growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro-cultured mammalian cells at sub-physiological temperatures. Mol. Biotechnol. 39:69-77.

162

Onadipe AO, Metcalfe HK, Freeman PR, James C (2001) Capillary-aided cell cloning. In: Animal Cell Technology: from Target to Market. Eds: Lindner-Olsson E et al. Kluwer Academic Publishers pp72-74. ♦ A method for one-step cloning with a high probability of monoclonality is

described.

112.

Peakman TC , Worden J, Harris RH, Cooper H, Tite J, Page MJ, Gewert DR, Bartholemew M, Crowe JS, Brett S (1994) Comparison of expression of a humanized monoclonal antibody in mouse NS0 myeloma cells and Chinese hamster ovary cells. Hum. Antibod. Hybrid. 5:65-74. ♦ Compared DHFR-CHO and GS-NS0 for expression of an IgG1. Monitored copy

number and steady state mRNA levels during selection and amplification for both systems. In NS0 and CHO cells making equivalent amounts of antibody, the copy number was lower in NS0 although heavy chain mRNA levels were virtually identical. Antibody purified from both systems behaved identically in a number of functional assays.

56.

Porter AJ, Dickson AJ, Racher AJ (2010) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors. Biotechnol. Prog. 26:1446-1454.

189.

Porter AJ , Racher A, Preziosi R, Dickson AJ (2010) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation. Biotechnol. Bioeng. 26:1455-1464.

190.

Tait AS , Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC (2004) Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol. Bioeng. 88:707-721. ♦ Describes use of polyethyleneimine-mediated transfection as a method for the

very rapid production of milligramme quantities of antibody.

143.

Underhill MF , Smales CM, Naylor LH, Birch JR, James DC (2007) Transient gene expression levels from multigene expression vectors. Biotechnol. Prog. 23:435-443. ♦ Authors evaluated use of IRES elements in GS expression vectors for

expression of multimeric proteins. Overall, it appeared that relative protein expression levels expected from heterologous gene products in a multigene vector could not be predicted on copy number alone and it is important to

137.


characterize multigene or oligocistronic systems prior to use. Underhill MF, Birch JR, Smales CM, Naylor LH (2005) eIF2α phosphorylation, stress perception, and the shutdown of global protein synthesis in cultured CHO cells. Biotechnol. Bioeng. 89:805-814. ♦ Used GS-CHO expressing TIMP-1 as a model system to investigate relationship

between cellular protein synthesis, cellular perception of stress, translation attenuation and specific antibody production rate.

138

Wilson RH (1993) Glutamine synthetase gene amplification in Chinese hamster ovary cells. In: "Gene Amplification in Mammalian Cells" (ed.) Kellens RE, Marcel Dekker Inc (New York) pp 301-311. ♦ Review of GS properties, its amplification in CHO cells using methionine

sulphoximine and its cloning and use as a selectable marker.

50.

Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF (2009) High-level protein expression in scalable CHO transient transfection. Biotechnol. Bioeng. 103:542-551. ♦ Describes development of transient expression system using polyethyleneimine

for GS-CHO. Achieved antibody concentrations up-to 80 mg/L. Saw similar product characteristics for the same antibody expressed in both transient and stably transfected cells. Differences were seen between antibody expressed transiently in HEK293 cells compared to stable GS-CHO cell lines.

176.

Ye J, Alvin K, Latif H, Hsu A, Parikh V, Whitmer T, Tellers M, de la Cruz Edmonds MC, Ly J, Salmon P, Markusen JF (2010) Rapid protein production using CHO stable transfection pools. Biotechnol. Prog. 1431-1437.

194.

SECTION 2: Cell line Stability

Bailey LA , Hatton D, Field RP, Dickson AJ (2012) Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture. Biotechnol. Bioeng. 109:2093-2103.

210.

Barnes LM, Bentley CM, Dickson AJ (2004) Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells. Biotechnol. Bioeng. 85:115 – 121. ♦ Following their 2003 paper Barnes et al have focused in on a number of NS0

cell lines, both stable and unstable for recombinant gene expression in long term culture. No change in transfected gene copy number is associated with declining productivity in unstable cell lines. However, levels of mRNA once below a critical “threshold point” appears to indicate a cell line whose productivity will decline. The authors hypothesise that genomic responses to disturbances in the genomic environment (possibly the result of vector integration) may be responsible. A number of solutions are proposed.

115.

Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production from recombinant mammalian cells. Biotechnol. Bioeng. 81:631 – 639. ♦ This paper provided a comprehensive review of stability issues related to

recombinant protein expression. Instability in hybrodomas, DHFR-CHOs and

116.


GS-NS0s is discussed. On the whole GS-NS0 cell lines used for manufacturing are stable. Any unstable cell lines created are usually discarded early on, for example, during the selection stage of cell line creation. Prospective molecular mechanisms for instability are discussed and approaches to improve expression levels and ensuring stability of production are also presented.

Barnes LM, Bentley CM, Dickson AJ (2003) Stability of recombinant protein production in the GS-NS0 expression system is unaffected by cryopreservation. Biotechnol. Prog. 19:233 – 237. ♦ Study shows that cryopreservation and revival procedures do not alter the

stability characteristics of GS-NS0 cell lines.

117.

Barnes LM, Bentley CM, Dickson AJ (2001) Characterisation of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnol. Bioeng. 73:261-270. ♦ Selection of production clones on the basis of growth and productivity alone will

not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.

142.

Bird P, Bolam E, Castell L., Obeid O, Darton N, Hale G (1998) Glutamine synthetase transfected cells may avoid selection by releasing glutamine. In: New Developments and New Applications in Animal Cell Technology. Eds: Merten OW, Perrin P, Griffiths B Kluwer Acad. Publishers, pp43-49. ♦ Noted that in some circumstances, particularly hollow fibre systems, GS

tranfected cell lines may release sufficient glutamine to overcome the selection pressure imposed by glutamine free media.

87.

Cosgrove L, Lovrecz GO, Verkuylen A, Cavaleri L, Black LA, Bentley JD, Howlett GJ, Gray PP, Ward CW, McKern NM (1995) Purification and properties of insulin receptor ectodomain from large scale mammalian cell culture. Protein Expression and Purification. 6:789-798. ♦ Ectodomain expressed in GS-CHO and cells grown in a perfused 40 litre airlift

fermenter on microcarrier beads. MSX required to maintain stable production (amplified cell line) but was omitted from production fermenter.

78.

Dorai H , Corisdeo S, Ellis D, Kinney C, Chomo M, Hawley-Nelson P, Moore G, Betenbaugh MJ, Ganguly S (2012) Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins. Biotechnol. Bioeng. 109:1016-1030.

208.

de la Cruz Edmonds MC , Tellers M, Chan C, Salmon P, Robinson DK, Markusen JF. (2006) Development of transfection and high-producer screening protocols for the CHOK1SV cell system. Mol. Biotechnol. 34:179-190.

175.

Guerini D, Schroder S, Foletti D, Carafoli E (1995) Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane

Ca2+ ATPase. J. Biol. Chem. 270:14643-14650.

67.


♦ GS–CHO cell lines created. Initial transfectants were subjected to additional

rounds of selections with MSX resulting in clones with 4 to 10 gene copies per cell. The stability of expression of transfected cells was examined in the presence and absence of selective drug (MSX). In the absence of MSX productivity was retained over short culture periods but was substantially decreased after 30 to 40 passages. In contrast stable production was maintained for 6 months in the presence of MSX.

Hassell T, Brand H, Renner G, Westlake A, Field RP (1992) Stability of production of recombinant antibodies from glutamine synthetase amplified CHO and NS0 cell lines. In: Animal Cell Technology. Developments Processes and Products, Butterworths, pp42-47. ♦ Examines stability of amplified and non amplified cell lines. Yield of 895 mg/l

achieved for a GS-NS0 cell line in serum-free fed-batch antibody culture.

30.

He L, Winterrowd C, Kadura I, Frye C (2012) Transgene copy number distribution profiles in recombinant CHO cell lines revealed by single cell analyses. Biotechnol. Bioeng. 109:1713-1722.

211.

Kearns B, Lindsay D, Manahan M, McDowall J, Rendeiro D (2003) NS0 batch cell culture process characterisation: a case study. BioProcessing Journal Jan/Feb 52-57. ♦ Investigation into decline in productivity as cell culture generation number

increased. No genetic instability seen but a phenotypic instability, related to the metabolic state of the culture, is reported.

110.

Kim M , O'Callaghan PM, Droms KA, James DC (2011) A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol. Bioeng. 108:

204.

Laubach VE, Garvey EP, Sherman PA (1996) High-level expression of human inducible nitric oxide synthase in Chinese hamster ovary cells and characterisation of the purified enzyme. Biochem. Biophys. Res. Comm. 218:802-807. ♦ iNOS expressed in CHO cells using GS system. Amplification in the presence

of 400µm MSX increased product levels 3 to 4 fold. Expression was enhanced by sodium butyrate (2mM). Expression levels were at least 20-fold higher than those reported for a baculovirus expression system. Also noted that sodium butyrate could restore high level expression in some cell lines in which iNOS expression declined with time in culture.

88.

Porter AJ, Dickson AJ, Barnes LM, Racher AJ (2007) Antibody production by GS-CHO cell lines over extended culture periods. In: Cell technology for Cell Products (Ed: Smith R) Springer, Dordrecht, pp137-140.

157.

Racher AJ (2005) Stability and Suitability of GS-NS0 Cell Lines for Manufacturing Antibodies. BioProcessing Journal.4:61-65.

124.

SECTION 3: Antibody Production

Alete DE, Racher AJ, Birch JR, James DC, Smales CM (2005) The functional competence of animal cells: analysis of the secretory pathway. In: Animal Cell Technology meets Genomics (Eds: Gòdia F, Fussenegger,M), Springer, Dordrecht,

129.


pp71-74. Alete DE, Racher AJ, Birch JR, Stansfield SH, James DC, Smales CM (2005) Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities. Proteomics 5:4689-4704.

122.

Bebbington CR (1991) Expression of antibody genes in non lymphoid mammalian cells. Methods in Enzymol. 2:136-145. ♦ Describes GS vector for use in CHO cells and antibody production in excess of

200mg/L.

1.

Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnol. 10:169-175. ♦ Describes use of GS selection system in NS0 for production of recombinant

antibody (cB72.3) to yields of 560 mg/L in fed batch airlift.

3.

Bebbington CR (1995) Expression of antibody genes in mammalian cells. In: "Monoclonal antibodies: the next generation", Zola, H. (ed.) Bios Scientific (Oxford) pp165-181. ♦ Review of expression systems including GS. Also discusses glycosylation of

antibodies.

71.

Bell SL, Bebbington CR, Bushell ME, Sanders PG, Scott MF, Spier RE, Wardell JN (1991) Genetic engineering of cellular physiology. In: Production of Biologicals from Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp304-306. ♦ GS gene used to confer glutamine independence on a hybridoma.

4.

Bell SL, Bushell ME, Scott MF, Wardell JN, Spier RE, Sanders PG (1992) Genetic modification of hybridoma glutamine metabolism: physiological consequences. In: Animal Cell Technology: Developments, Processes and Products. Eds: Spier RE, Griffiths JB, MacDonald C. Butterworth-Heinemann, pp 180-182. ♦ GS gene transfected into hybridoma cell to confer glutamine independence.

5.

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushell ME, Sanders PG (1995) Genetic engineering of hybridoma glutamine metabolism. Enzyme and Microbial Technol. 17:98-106. ♦ GS gene used to confer glutamine independence on a hybridoma.

65.

Bentley KJ, Gewert R, Harris WJ (1998) Differential efficiency of expression of humanized antibodies in transient transfected mammalian cells. Hybridoma 17:559-567. ♦ Used GS vectors to examine factors influencing level of transient expression of

antibodies in CHO cells. Expression levels greatly influenced by amino acid sequence of variable regions and by different combinations of antibody heavy

106.


and light chains. The authors note that it was possible to obtain stable transfectants that overcame the limitations observed in transient expression.

Beyer T. Lohse S, Berger S, Peipp M, Valerius T, Dechant M (2009) Serum-free production and purification of chimeric IgA antibodies. J Immunol Meth 346:26-37. ♦ Describes use of GS-CHO cell lines to express an IgA molecule.

181.

Bi J -X, Shuttleworth J, Al-Rubeai M (2004) Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells. Biotechnol. Bioeng. 85:741-749.

150.

Bibila TA, Ranucci CS, Glazomitsky K, Buckland BC, Aunins JG (1994) Monoclonal antibody process development using medium concentrates. Biotechnol. Prog. 10:87-96. ♦ Describes fed-batch process using concentrated medium with GS-NS0 cell lines

making recombinant antibodies. Up to 7-fold increases in antibody titre were achieved compared with batch culture.

54.

Bibila TA , Robinson DK (1995) In pursuit of the optimal fed-batch process for monoclonal antibody production. Biotechnol. Prog. 11:1-13. ♦ A review of approaches taken to optimise fed-batch processes for antibody

production.

60.

Birch JR, Bebbington CR, Field R, Renner G, Brand H, Finney H (1993) The production of recombinant antibodies using the glutamine synthetase (GS) system. In: Animal Cell Technology: Basic and Applied Aspects 5:573-577. ♦ Review of GS system with examples of fermenter productivity and process

improvement.

6.

Birch JR , Froud S (1994) Mammalian cell culture systems for recombinant protein production. Biologicals 22:127-133. ♦ Gives example of Mab production (200 mg/L) using GS-NS0 cells in protein-free

medium.

53.

Birch JR , Mainwaring DO, Racher AJ (2005) Use of the Glutamine Synthetase (GS) Expression System for the rapid development of highly productive mammalian cell processes. In: Modern Biopharmaceuticals (Knäblein, J. ed), WILEY-VCH Verlag GmbH & Co KGaA, pp809-832. ♦ Status of GS Gene Expression System, Summer of 2004.

131.

Birch JR , Racher AJ (2006) Antibody production. Advanced Drug Delivery Reviews 58:671-685. ♦ General review of antibody production systems, including the GS Gene

Expression System.

130.

Bird P, Bolam E, Castell L, Obeid O, Darton N, Hale G (1998) Glutamine synthetase transfected cells may avoid selection by releasing glutamine. In: New Developments and New Application in Animal Cell Technology. Eds: Merten OW, Perrin P, Griffiths B. Kluwer Acad. Publishers, pp43-49.

87.


♦ Noted that in some circumstances, particularly hollow fibre systems, GS

tranfected cell lines may release sufficient glutamine to overcome the selection pressure imposed by glutamine free media.

Bloom JW, Madanat MS, Marriott D, Wong T, Chan SY. (1997) Intrachain disulfide bond in the core hinge region of human IgG4. Protein Sci. 6:407-415.

217.

Brand HN, Froud SJ, Metcalfe HK, Onadipe AO, Shaw A, Westlake AJ (1994) Selection strategies for highly productive recombinant cell lines. In: Animal Cell Technology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, Griffiths JB, Belthold W, Butterworth-Heinemann, 55-60. ♦ Compares productivity of attached and suspended GS-CHO. Reviews criteria

for selecting highly productive GS-NS0 and GS-CHO cell lines.

7.

Broad D, Boraston R, Rhodes M (1991) Production of recombinant proteins in serum-free media. Cytotechnol. 5:47-55. ♦ Compares productivity of three amplified GS-CHO lines making recombinant

proteins in the presence and absence of serum (no significant differences seen).

8.

Brown ME, Renner G, Field RP, Hassell T (1992) Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system. Cytotechnol. 9:231-236. ♦ Describes improvements in batch process leading to antibody yields ranging up

to 1 g/L. Serum-free medium used.

9.

Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PWHI, Sawyer LSW, Hendry RM, Dunlop N, Nara PL., Lanacchia M, Garratty E, Stiehm ER, Bryson YJ, Cao Y, Moore JP, Ho DD, Barbas CF (1994) Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266:1024-1027. ♦ A recombinant human antibody to virus envelope protein gp120 was expressed

in GS-CHO cells using methionine sulphoximine amplification. The whole antibody was constructed from a FAb fragment generated from a combinatorial phage display library.

55.

Charaniya S, Karypis G, Hu W-S (2009) Mining transcriptome data for function-trait relationship of hyper productivity of recombinant antibody. Biotechnol, Bioeng. 102:1654-1669.

172.

Davies SL, O'Callaghan PM, McLeod J, Pybus LP, Sung YH, Rance J, Wilkinson SJ, Racher AJ, Young RJ, James DC (2011) Impact of gene vector design on the control of recombinant monoclonal antibody production by Chinese hamster ovary cells. Biotechnol. Prog. 27:1689-1699.

203.

Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC. (2013) Functional heterogeneity and heritability in CHO cell populations. Biotechnol. Bioeng. 110:260-274.

218.

Dempsey J, Ruddock S, Osborne M, Ridley A. Sturt S, Field R (2003) Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase. Biotechnol. Prog. 19:175-178.

109.


♦ Repeated nutrient analysis and re-supplementation of serum-free antibody

producing NS0 cultures performed. As a result media and feeds were developed which gave 10-fold increases in antibody harvest titres up to 600mg/l.

Dinnis DM, Stansfield SH, Schlatter S; Smales CM; Alete D; Birch JR; Racher AJ; Marshall CT; Nielsen LK.; James DC (2006) Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol. Bioeng. 94:830-841.

133.

Dorai,H , Sauerwald,TM, Campbell A, Kyung Y-S, Goldstein J, Magill A, Lewis MJ, Tang QM, Jan D, Ganguly S, Moore G (2007) Investigation of protein microheterogeneity. A case study in rapid detection of mutation in mammalian production cell lines. BioProcess Intl 5(8):66-72. ♦ Describes usage of a variety of analytical techniques to characterise an

antibody-peptide fusion protein and identify a mutation that occurred post-transfection. Paper also includes data on GS vector copy number in two cell lines derived from CHOK1SV.

161

Downham MR, Farrell WE, Jenkins HA (1996) Endoplasmic reticulum protein expression in recombinant NS0 myelomas grown in batch culture. Biotechnol. and Bioengineer. 51:691–696. ♦ Production of ER proteins (GRP78/BiP, GRP94 and Erp72) increased during

decline phase of batch culture of GS-NS0 coincident with an increase in production rate of recombinant antibody and reduction in uptake of glucose and glutamate.

83.

Field RP, Brand H, Renner GL, Robertson HA, Boraston R (1991) Production of a chimeric antibody for tumour imaging and therapy from Chinese hamster ovary (CHO) and myeloma cells. In: Production of Biologicals from Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp742-744. ♦ Early work on expression of chimeric antibody (cB72.3) in GS-NS0. Achieved

240mg/l in unfed, serum-free fed-batch culture. An alternative expression system using neo and gpt genes as selectable markers in CHO cells gave 90mg/l.

16.

Fries S, Glazomitsky K, Woods A, Forrest G, Hsu A, Olewinski R, Robinson DK, Chartrain M (2005) Evaluation of disposable bioreactors. Rapid production of recombinant proteins by several animal cells. BioProcess International 3( Supp 6):36-44. ♦ Describes growth of antibody-producing GS-NS0 cell line in cell culture-bag

disposable bioreactor system.

155.

Froud SJ (1994) Selection strategies for highly productive recombinant lines. ♦ Paper read at 'Commercializing human monoclonal antibodies' conference. San

Diego, Feb 7-8 1994. Describes strategies for isolating cell lines making up to 1300mg of recombinant antibody/litre.

18.

Galbraith DJ, Tait AS; Racher AJ, Birch JR, James DC (2006) Control of culture 134.


environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnol. Prog. 22:753-762. ♦ Describes optimisation of polyethyleneimine-mediated transfection as a method

for the very rapid production of milligramme quantities of antibody. Hassell T, Brand H, Renner G, Westlake A, Field RP (1992) Stability of production of recombinant antibodies from glutamine synthetase amplified CHO and NS0 cell lines. In: Animal Cell Technology. Developments Processes and Products, Butterworths, pp42-47. ♦ Examines stability of amplified and non amplified cell lines. Yield of 895 mg/l

achieved for a GS-NS0 cell line in serum-free fed-batch antibody culture.

30.

Hayes NVL, Smales CM, Klappa P (2010) Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines. Biotechnol. Bioeng. 105:770-779.

186.

Hermes PA , Castro CD (2010) A fully defined, fed-batch, recombinant NS0 culture process for monoclonal antibody production. Biotechnol. Prog. 26:1411-1416.

193.

Ho Y, Varley J, Mantalaris A (2006) Development and analysis of a mathematical model for antibody-producing GS-NS0 cells under normal and hyperosmotic culture conditions. Biotechnol. Prog. 22:1560-1569. ♦ Describes a hybrid model, consisting of both unstructured and structured

elements, has been developed to describe cell growth and death, metabolism, and antibody production in the GS-NS0 system under normal culture conditions. The specific transcription and translation rates of heavy and light immunoglobulin chains were identified as parameters with the largest impact on the antibody production process.

127.

Ho Y, Kiparissides A, Pistikopoulos EN, Mantalaris A. (2012) Computational approach for understanding and improving GS-NS0 antibody production under hyperosmotic conditions. J. Bioscience Bioeng. 113:88-98.

222.

Ibarra N, Watanabe S, Bi JX, Shuttleworth J, Al-Rubeai M (2003) Modulation of cell cycle for enhancement of antibody production in perfusion culture of NS0 cells. Biotechnol. Prog. 19:224-228. ♦ NS0 cells metabolically engineered to express cytostatic and anti-apoptotic

genes. Resulting cell line exhibited a 4-fold increase in productivity in the arrested phase compared to the proliferative phase.

111.

Kadarusman J , Bhatia R, McLaughlin J, Lin WR (2005) Growing cholesterol-dependent NS0 myeloma cell line in the Wave bioreactor system: overcoming cholesterol-polymer interaction by using pretreated polymer or inert fluorinated ethylene propylene. Biotechnol. Prog. 21:1341-1346. ♦ Discusses choice of material for manufacture of disposable bioreactor systems

168.

Keen MJ, Hale C (1996) The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker. Cytotechnol. 18:207-217.

80.


♦ Describe protein-free medium for GS-NS0 supplemented with cholesterol, phosphatidylcholine and β-cyclodextrin. Adapted cells to become cholesterol independent.

Khoo SHG, Al -Rubeai M (2009) Detailed understanding of enhanced specific antibody productivity in NS0 myeloma cells. Biotechnol. Bioeng. 102:188-189.

171.

Kilgore BR , Lucka A, Patel R, Andrien B, Dhume ST (2005) Cell line Switch from NS0 to CHO Causes Several Changes in Glycosylation of Recombinant IgG. ACS 33rd Northeast Regional Meeting (Abstract) ♦ http://acs.confex.com/acs/nerm05/techprogram/P21476.HTM

214.

King DJ, Byron OD, Mountain A, Weir N, Harvey A, Lawson ADG, Proudfoot KA, Baldock D, Harding SE, Yarranton GT, Owens RJ (1993) Expression, purification and characterization of B72.3 Fv fragments. Biochem. J. 290:723-729. ♦ Fv expressed in GS-CHO and in E. coli. Yields of 4 mg/L achieved in CHO roller

bottles and 450 mg/L in E. coli fermentations.

32.

Lifely MR, Hale C, Boyce S, Keen MJ, Phillips J (1995) Glycosylation and biological activity of Campath - IH expressed in different cell lines and grown under different culture conditions. Glycobiol. 5:813-822. ♦ Antibody expressed in CHO (DHFR), rat YO myeloma and NS0 (GS) cells.

Glycosylation varied significantly between cell lines and to a minor extent depending on culture conditions. YO antibody was unusual in having a bisecting Glc NAc on the core oligoscaccharide and NS0 antibody appeared to be under glycosylated. YO antibody had enhanced activitity in ADCC assays compared with CHO or NS0.

74.

Lewis AP, Parry N, Peakman TC, Scott-Crowe J (1992) Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies. Hum. Antibod. Hybrid. 3:146-152. ♦ Human Ig genes rescued from human hybridoma cells and expressed in rat YO

cells using GS vector.

34.

Lohse S , Derer S, Beyer T, Klausz K, Peipp M, Leusen JH, van de Winkel JG, Dechant M, Valerius T (2011) Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumour cell killing. J. Immunol. 186:3770-3778.

205.

Mason M , Sweeney,B. Cain K, Stephens P, Sharfstein ST (2012) Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in chinese hamster ovary cells. Biotechnol. Prog. 28:846-855.

213.

McKenna SL , Cotter TG (2000) Inhibition of caspase activity delays apoptosis in a transfected NS0 myeloma cell line. Biotech. Bioeng. 67:165-176. ♦ Z-VAD-fmk, a specific inhibitor of caspases reduced apoptosis in GS-NS0 cells

but did not increase productivity. The inhibitor did not prevent mitochondrial dysfunction.

102.

McLeod J , O'Callaghan PM, Pybus LP, Wilkinson SJ, Root T, Racher AJ, James DC (2011) An empirical modeling platform to evaluate the relative control discrete

197.


CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer. Biotechnol. Bioeng. 108:2193-2204. Mead EJ, Chiverton LM, Spurgeon SK, Martin EB, Montague GA, Smales CM, von der Haar T. (2012) Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines. PLOS One 7(10):e47422. ♦ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468484/

221.

Moran EB, McGowan ST, McGuire JM, Frankland JE, Oyebade IA, Waller W, Archer LC, Morris LO, Pandya J, Nathan SR, Smith L., Cadette ML, Michalowski JT (2000) A systematic approach to the validation of process control parameters for monoclonal antibody production in fed-batch culture of a murine myeloma. Biotechnol. Bioeng. 69:242-255. ♦ Describes an approach to validating control parameters for a GS-NS0 cell line.

105.

O'Callaghan PM, McLeod J, Pybus LP, Lovelady CS, Wilkinson SJ, Racher AJ, Porter AJ, James DC (2010) Cell line-specific control of recombinant monoclonal antibody production by CHO cells. Biotechnol. Bioeng. 106:938-951.

191.

O'Connor KC , Muhitch JW, Lacks DJ, Al-Rubeai M (2006) Modelling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells. Biotechnol. Lett. 28:1919-1924.

154.

Paterson T, Innes J, McMillan L, Downing I, McCann Carter MC (1998) Variation in IgG1 heavy chain allotype does not contribute to differences in biological activity of two human anti-Rhesus (D) monoclonal antibodies. Immunotechnol. 4:37-47. ♦ Two anti-Rhesus (D) antibodies ‘rescued’ from heterohybrid cell lines and re-

expressed in NS0 cells using GS selection. Secretion levels were not improved by amplification. The two recombinant antibodies exhibited identical in vitro properties to the parent bodies.

97.

Peakman TC, Worden J, Harris RH, Cooper H, Tite J, Page MJ, Gewert DR, Bartholemew M, Crowe JS, Brett S (1994) Comparison of expression of a humanized monoclonal antibody in mouse NS0 myeloma cells and Chinese hamster ovary cells. Hum. Antibod. Hybrid. 5:65-74. ♦ Compared CHO DHFR and GS NS0 for expression of an IgG1. Monitored copy

number and steady state mRNA levels during selection and amplification for both systems. In NS0 and CHO making equivalent amounts of antibody, the copy number was lower in NS0 although heavy chain mRNA levels were virtually identical. Antibody purified from both systems behaved identically in a number of functional assays.

56.

Porter AJ , Mohindra A, Porter JM, Racher AJ (2011) Does earlier use of productivity enhancers during cell line selection lead to the identification of more productive cell lines? BMC Proceedings 5(Suppl8):9. ♦ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284934/?tool=pubmed

209.

Reid CQ, Tait AS, Baldascini H, Mohindra A, Racher AJ, Bilsborough S, Smales CM, Hoare M (2010) Rapid whole monoclonal antibody analysis by mass spectrometry: An ultra scale-down study of the effect of harvesting by centrifugation

188.


on the post-translational modification profile. Biotechnol. Bioeng. 107:85-95. Rendall MH, Maxwell A, Tatham D, Khan P, Gay RD, Kallmeier RC, Wayte JRT, Racher AJ (2005) Transfection to manufacturing: reducing timelines for high yielding GS-CHO processes. In: Animal Cell Technology meets Genomics (Eds: Gòdia F, Fussenegger M), Springer, Dordrecht, pp701-704.

135.

Robinson DK, Chan CP, Yu Ip CC, Seamans TC, Lee DK, Lenny AB, Tung JS, DiStefano DJ, Munshi S, Gould SL, Tsai PK, Irwin J, Mark GE, Silberklang M. (1994) Product consistency during long-term fed-batch culture. In: Animal Cell Technology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, Griffiths JB, Berthold W. Butterworth-Heinemann, pp763-767. ♦ Expressing antibody genes in GS-NS0. Yields up to 1.8g/l in three to four week

duration fed-batch. Glycoform pattern shifts towards incomplete mannose-terminated glycans as culture progresses.

46.

Robinson DK, Chan C., Yu Ip C, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J, Silberklang M (1994) Characterization of a recombinant antibody produced in the course of a high yield fed-batch process. Biotechnol. Bioeng. 44:727-735. ♦ Anti-HIV gp120 MAb expressed in non-amplified GS-NS0 system. Fed-batch

process developed which increased yields ten-fold compared with batch culture. The clone produced 0.86g MAb/l over 22 days. Antigen binding and MWt remained constant throughout culture. Four major IEF bands were observed with a minor fifth more acidic band appearing later in culture. Pattern of glycoforms secreted by cells changes as culture progresses.

52.

Robinson DK, Seamans TC, Gould SL, DiStefano DJ, Chan CP, Lee DK, Bibila T, Glazomitsky K, Munshi S, Daugherty B, O'Neill Palladino L, Stafford-Hollis J, Hollis GF, Silberklang M (1994) Optimization of a fed-batch process for production of a recombinant antibody. Reprinted from Biochem. Engineering VIII, Ann. (NY) Acad. Sci. 745:285-296. ♦ Describes the development of an optimized fed-batch process for a GS-NS0 cell

line secreting a monoclonal antibody. Includes data on amino acid consumption rates.

62.

Robinson DK, DiStefano D, Gould SL, Cuca G, Seamans TC, Benincasa D, Munshi S, Chan CP, Stafford-Hollis J, Hollis GF, Jain D, Ramasubramanyan K, Mark GE, Silberklang M (1995) Production of engineered antibodies in myeloma and hybridoma cells. In: Antibod. Engineering. Eds: Wang H, Imanaka T pp1 - 14, Worthington: ACS. ♦ Detailed description of production of recombinant antibody in a GS-NS0 system

for production of a recombinant antibody.

84.

Schenerman M A, Hope JN, Kletke C, Singh JK, Kimura R, Tsao EI, Folena-Wasserman G (1999) Comparability Testing of a Humanized Monoclonal Antibody (Synagis®) to Support Cell Line Stability, Process Validation and Scale-Up for Manufacturing. Biologicals. 27:203-215.

126.

Schlatter S, Stansfield SH, Dinnis DM, Racher Andrew J, Birch John R, David C James (2005) On the Optimal Ratio of Heavy to Light Chain Genes for Efficient

121.


Recombinant Antibody Production by CHO Cells. Biotechnol. Prog. 21(1):122-133. Seamans TC, Gould SL, DiStefano DJ, Silberklang M, Robinson DK (1994) Use of lipid emulsions as nutritional supplements in mammalian cell culture. Ann. (NY) Acad. Sci. 745:240-243. ♦ Describes preparation of stable lipid emulsions to substitute for lipoproteins in

the culture of NS0 cells secreting a recombinant antibody.

61.

Seamans TC, Fries S, Beck A, Wurch T., Chenu S, Chan C, Ushio M, Bailey J, Kejariwal A, Ranucci C, Villani N, Ozuna S, Goetsch L, Corvaia N, Salmon P, Robinson DK, Chartrain M (2008) Cell cultivation process transfer and scale-up in support of production of early clinical supplies of an anti IGF-1R antibody, Part 1. BioProcess International 6(3):26-36.

163.

Seamans TC, Fries S, Beck A, Wurch T., Chenu S, Chan C, Ushio M, Bailey J, Kejariwal A, Ranucci C, Villani N, Ozuna S, Goetsch L, Corvaia N, Salmon P, Robinson DK, Chartrain M (2008) Cell cultivation pocess transfer and scale-up in support of production of early clinical supplies of an anti IGF-1R antibody, Part 2. BioProcess International 6(4):34-42.

164.

Sellick CA , Croxford AS, Maqsood AR, Stephens G, Westerhoff HV, Dickson (2011) Metabolite profiling of recombinant CHO cells: Designing tailored feeding regimes that enhance recombinant antibody production. Biotechnol. Bioeng. 108:3025-3031.

212.

Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ, Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol. Bioeng. 88:474–488. ♦ Proteomic analysis of four different NS0 cell lines, with different levels of

antibody production, identified a number of proteins with altered abundances. Amongst these proteins a number of molecular chaperones involved in folding and assembly of immunoglobulins were identified. The authors also report an abundance of light chain protein over heavy chain protein a likely prerequisite for efficient MAb production.

118.

Stephens S, Emtage S, Vetterlein O, Chaplin L., Bebbington CR, Nesbitt A, Sopwith M, Athwal D, Novak C, Bodmer M (1995) Comprehensive pharmacokinetics of a humanised antibody and analysis of residual anti-idiotypic responses. Immunol. 85:668-674. ♦ Describes pharmacokinetics in monkeys and human volunteers of anti TNFα

antibody (CDP571) produced in GS-NS0. In humans the antibody was well tolerated with a half life of approx. 13 days and anti CDP571 antibodies were low or undetectable at higher doses. At lower doses there was a transient IgM response which was anti-idiotypic.

72.

Tait AS , Hogwood CEM, Smales CM, Bracewell DG (2012) Host cell protein dynamics in the supernatant of a mAb producing CHO cell line. Biotechnol. Bioeng. 109:971-982.

206.

Teixeira AP , Duarte TM, Carrondo MJT, Alves PM (2011) Synchronous fluorescence spectroscopy as a novel tool to enable PAT applications in bioprocesses. Biotechnol. Bioeng. 108:1852-1861.

198.


Tey BT, Singh RP, Al-Rubeai M (1999) Influence of bcl-2 over-expression on NS0 and CHO culture viability and chimeric antibody productivity. In: Animal Cell Technology: Products from Cells, Cells as Products. Eds: Bernard A et al. pp 59-61 Kluwer. ♦ Over-expression of bcl-2 significantly reduced the rate of cell death in a GS-

CHO and in GS-NS0 cell line and resulted in a 19% and 25% increase, respectively in antibody production.

98.

Tey BT, Singh RP, Piredda L., Piacentini M, Al-Rubeai M (2000) Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotech. Bioeng. 68:31-43. ♦ Transfection with the control expression vector (Neo marker) used in this study

and exposure to the selective drug G418 led to upregulation of endogenous bcl-2. This led to an increase in cell viability in cell cultures and prolonged survival. There was no influence on antibody titre.

99.

Tey BT , Singh RP, Piredda L, Piacentini MP, Al-Rubeai M (2000) Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures. J. Biotechnol. 79:147-159. ♦ In batch culture, no difference was seen in product concentration between cell

line over-expressing Bcl-2 and control cell line. However, in fed-batch culture using a concentrated amino acid feed, antibody concentration was increased 60% in the Bcl-2 over-expressing cell line..

147.

Tey BT , Al-Rubeai M. (2004) Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity. Apoptosis 9:843-852.

160

Tey BT , Al-Rubeai M (2005) Effect of Bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells. J Biosci Bioeng 100:303-310.

159

van Berkel PHC , Gerritsen J, Perdok G, Valbjørn J, Vink T, van de Winkel JGJ, Parren PWHI. (2009) N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG. Biotechnol. Prog. 25:244-251.

174.

Wayte J, Boraston R, Bland H, Varley J, Brown M (1997) pH: effects on growth and productivity of cell lines producing monoclonal antibodies: control in large-scale fermenters. The Genetic Engineer and Biotechnol. 17:125-132. ♦ Describes effect of pH on growth and productivity of a GS-NS0 cell line making

a humanised monoclonal antibody. Productivity increased at pH7.1 compared with 7.4.

94.

Yoon S , Konstantinov KB (1994) Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors. Biotech. Bioeng. 44:983-990. ♦ Describes oxygen uptake monitoring for cultures of GS-NS0 cells making

humanised anti TNF antibody. Used a perfused stirred reactor with cell retention device. OUR was in the range 7 to 13 pg/cell per hour depending on cell density.

59.


Watanabe S , Shuttleworth J, Al-Rubeai M (2002) Regulation of cell cycle and productivity in NS0 cells by the over-expression of p21CIP1. Biotechnol. Bioeng. 77:1-7.

148.

Wu MH, Dimopoulos G, Mantalaris A, Varley J. (2004) The effect of hyperosmotic pressure on antibody production and gene expression in the GS-NS0 cell line. Biotechnol. Appl. Biochem. 40:41-46. ♦ Describes growth and productivity kinetics as well as gene expression, using

microarray technology, at various osmolalities..

128.

Zhou W, Chen C-C, Buckland B, Aunins J (1997) Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production. Biotechnol. Bioeng. 55:783-792. ♦ Describes feeding strategy leading to final antibody concentration in excess of

2.7g/L. Describes transitions in metabolism caused by nutrient depletion. The cell line used in the study had three vector copies.

90.

SECTION 4: Recombinant Proteins (non-antibody) from NS0 Cells

Cannon -Carlson S, Varnerin J, Tsarbopoulos A, Jenh C-H, Cox MA, Chou C-C, Connelly N, Zavody P, Tang JC-T (1998) Expression Purification and Characterisation of Recombinant Human Interleukin-13 from NS0 Cells. Protein Expression and Purification. 12:239-248. ♦ IL-13 expressed in NS0 cells using GS expression system. Cells grown in 15

litre fed batch culture. Details given of the medium and feed used. The protein is unglycosylated.

92.

Chen P, Cataldo S, Dennis P, Popoloski J, Dabora R (1994) A comparison of Chinese hamster ovary (CHO) and mouse myeloma (NS0) cell lines producing a recombinant protein. American Chem. Soc. Abs. of 207th National Meeting San Diego. ♦ Rec protein expressed at level of 120 mg/l with CHO DHFR in suspension and

at 450 mg/l in GS-NS0 in batch culture (following medium optimisation). Substantial differences seen in the carbohydrate structure and pharmacokinetic properties of the protein produced in the two cell lines.

11.

Flesher AR, Marzowski J, Wang W-C, Raff HV (1995) Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion protein: correlations of oligosaccharide content with in-vivo clearance profile. Biotech. and Bioeng. 46:399-407.

♦ Compared glycosylation and in-vivo clearance of a CTLA4/immunoglobulin

fusion protein made in GS-NS0 cells with that made in CHO cells. NS0 derived protein had no detectable N-acetylneuraminic acid and had concomitant accelerated clearance.

66.

Gofton CM, Roberts G, Bergin S, Owens RJ (1992) The rapid production of recombinant rabbit metalloproteinases in myeloma cells. In: Animal Cell Technology: Developments, processes and products. Butterworths, pp48-50.

23.


♦ Procollagenase, prostromelysin and TIMP expressed rapidly in NS0 using GS giving non-optimised fermenter yields of 46, 48.5 and 59.2 mg/l respectively.

Li SL, Liang SJ, Guo N, Wu AM, Fujita-Yamaguchi Y (2000) Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification and effect on tumor growth.

♦ Recloning of anti-IGF-IR monoclonal antibody as a single-chain Fv with successful expression obtained in a NS0 cell line.

113.

Murphy G, Houbrechts A., Cockett MI, Williamson RA, O'Shea M, Docherty AJP (1991a) The N-terminal domain of the tissue inhibitor of metalloproteinases (TIMP) is inhibitory. Biochem. 30:8097-8102. ♦ TIMP 1 and a truncated version expressed in GS-NS0.

41.

Murphy G, Cockett MI, Ward RV, Docherty AJ (1991b) Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and –2 and punctuated metalloproteinase (PUMP). Biochem. J. 277:277-279. ♦ Punctuated metalloproteinase (PUMP) and stromelysin 2 expressed in GS-NS0

cells.

40.

Murphy G, Allan JA, Willenbrock F, Cockett MI, O'Connell JP, Docherty AJ (1992a) The role of the C-terminal domain in collagenase and stromelysin specificity. J. Biol. Chem. 267:9612-9618. ♦ Collagenase, stromelysin and truncated versions of these enzymes expressed

in GS-NS0 cells.

39.

Murphy G, Willenbrock F, Ward RV, Cockett MI, Eaton D, Docherty AJP (1992b) The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases. Biochem. J. 283:637-641. ♦ Gelatinase and a variant expressed in GS-NS0.

42.

O Shea M, Willenbrock F, Williamson RA, Cockett MI, Freedman RB, Reynolds JJ, Docherty AJP , Murphy G (1992) Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: Importance of the N-terminal region between cysteine 3 and cysteine 13. Biochem. 31:10146-10152. ♦ Wild type and mutated TIMPS in GS-NS0 cells. Most produced at 30mg/l but for

some variants productivity very low.

43.

Robinson MK, Andrew D, Rosen H, Brown D, Ortlepp S, Stephens P, Butcher EC (1992) Antibody against the leu-CAM b-chain (CD18) promotes both LFA-1 and CR3-dependent adhesion events. J. Immunol. 148:1080-1085. ♦ CD18 expressed in GS-NS0 cells.

45.

Rossman C, Sharp N, Allen G, Gewert D (1996) Expression and purification of recombinant, glycosylated human interferon alpha 2b in murine Myeloma NS0 cells. Protein Expression and Purification. 7:335–342.

85.


♦ Expressed Interferon alpha in GS-NS0 cells and isolated a cell line expressing 20 µg/106 cells/24h and accumulating 120 µg/mL in culture. Glycosylation was similar to that of non recombinant interferon purified from human Namalwa cells. This is the highest reported level of glycosylated, recombinant IFN expression in a stable mammalian system.

Young RJ , Owens RJ, Mackay GA, Chan CMW, Shi J, Hide M, Francis DM, Henry AJ, Sutton BJ, Gould HJ (1995) Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants. Protein Eng. 8:193-199. ♦ Established permanent GS-CHO and GS-NS0 cell lines making Fc. The NS0

cell line accumulated up to 100 mg product per litre in contrast to ~2 mg/L in CHO.

107

Zhou W, Bibila T, Glazomitsky K, Montalvo J, Chan C, DiStefano D, Munshi S, Robinson D, Buckland B, Aunins J. (1996) Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NS0 cell cultures. Cytotechnol. 22:239-250. ♦ Transfected NS0 cells with GS vector containing growth hormone genes and

sequence for targeting integration by homologous recombination (for optimal expression). Operated fed-batch cultures at 250l scale and information on operating conditions is given. Yields of mouse and rat growth hormone were 580 and 240 mg/l respectively. Data presented on cell metabolism. Glucose consumption rate decreased during transition to stationary phase and lactate was consumed during stationary phase.

77.

SECTION 5: Recombinant Proteins (non-antibody) from CHO Cells

Blochberger TC, Cooper C, Peretz D, Tatzelt J, Griffith OH, Baldwin MA, Prusiner SB (1997) Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. Protein Engineering 10:1465-1473

♦ Describes expression of hamster prion proteins in CHO cells using the GS

system.

91.

Brady RL, Dodson EJ, Dodson GG, Lange G, Davis SJ, Williams AF, Barclay AN (1993) Crystal structure of domains 3 and 4 of rat CD4: Relation to the NH2- terminal domains, Science 260:979-983. ♦ Recombinant rat CD4 D3 and D4 expressed using GS.

96.

Brown MH , Barclay AN (1994) Expression of immunoglubulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis. Protein Engineering 7:515-521.

♦ Describes the use of GS-CHO cells to express proteins in which immunoglobulin

superfamily domains ( CD4d 3 + 4 ) are used as expression vehicles for the production of chimeric proteins containing other superfamily domains.

95.

Castro MG, Tomasec P, Morrison E, Murray CA, Hodge P, Blanning P, Linton E, Lowry PJ, Lowenstein PR (1995) Mitogenic effects and nuclear localisation of

63.


procorticotrophin-releasing hormone expressed within stably transfected fibroblast cells (CHO-K1). Molecular and Cellular Endocrinol. 107:17-27. ♦ Hormone expressed in GS-CHO cells. Cells expressing hormone had an

increased proliferation rate. Clark e S, Dillon J, Smith A, Sotheran E (2004) Strategies for producing commercial cell lines. BioProcess Interntl. 2(4) April 2004, pp 48 – 52. ♦ The cell culture experiences of Lorantis Ltd from Cambridge in the UK. They

chose the GS expression system and CHOK1 cells to construct a stable cell line expressing recombinant protein (Delta-1 derived from Notch binding protein) fused to an antibody constant region. Details of their project along methods for selection and cloning are presented.

114.

Classon BJ, Brown MH, Garnett D, Somoza C, Barclay AN, Willis AC, Williams AF (1992) The hinge regions of the CD8 alpha chain: structure, antigenicity and utility in expression of immunoglobulin superfamily domains. Int. Immunol. 4:(2), 215-225. ♦ Expression of SCD8α in GS-CHO. Yields of ca. 20mg/l in roller bottle culture.

Used 2mM sodium butyrate to enhance production.

12.

Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. BioTechnol. 8:662-667. ♦ Yields of 180mg/L achieved in shake flask culture. Sodium butyrate enhanced

production.

13.

Cockett MI, Bebbington CR, Yarranton GT (1991) The use of engineered EIA genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucl. Acids. Res. 19:319-325. ♦ TIMP expressed in GS-CHO with EIA. In the presence of EIA non amplified

lines gave levels comparable with gene amplified GS-CHO lines (110 µg/106

cells/24h). EIA also enhanced production of procollagenase (13 µg/106

cells/24h).

14.

Cosgrove L, Lovrecz GO, Verkuylen A, Cavaleri L., Black LA, Bentley JD, Howlett GJ, Gray PP, Ward CW, McKern NM (1995) Purification and properties of insulin receptor ectodomain from large scale mammalian cell culture. Protein Expression and Purification 6:789-798. ♦ Ectodomain expressed in GS-CHO and cells grown in a perfused 40 litre airlift

fermenter on microcarrier beads. MSX required to maintain stable production (amplified cell line) but was omitted from production fermenter.

78.

Crouch E, Chang D, Rust K, Pesson A, Heuser J (1994) Recombinant pulmonary surfactant protein D. J. Biol. Chem. 269:15808-15813 ♦ GS-CHO used to express rat SP-D.

64.

Davis SJ, Ward HA, Puklaver MJ, Willis AC, Williams AF, Barclay AN (1990) High level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants. J. Biol. Chem. 265:10410-10418.

15.


♦ S CD4 and S CD4 (half) expressed at levels of 80 and 25 µg/L from GS-CHO

cells. Used GMEM-S + 5% FCS and MSX (15 or 100 µM) in roller bottles. Sodium butyrate added to enhance production. Also used switch to serum free medium (GMEM-S + 2 mM butyrate) at final stage, to aid purification.

Dorai H, Nemeth JF, Cammaart E Wang Y, Tang QM, Magill A, Lewis MJ, Raju TS, Picha K, O'Neil K, Ganguly S, Moore G. (2009) Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODYTM: Factors that influence productivity and product quality. Biotechnol. Bioeng. 103:162-176.

173.

Fahnestock M, Johnson JL., Feldman RMR, Tsomides TJ, Mayer J, Narhi LO, Bjorkman PJ (1994) Effects of peptide length and composition on binding to an empty class 1 MHC heterodimer. Biochem. 33:8149-8158. ♦ Heterodimers expressed in GS CHO.

57.

Fahnestock ML, Johnson JL, Renny Feldman RM, Neveu JM, Lane WS, Bjorkman PJ (1995) The MHC class I homolog encoded by human cytomegalovirus binds endogenous peptides. Immunity 3:583-590. ♦ A secreted form of the MHC class I heavy chain homolog was expressed

together with human β2m using the GS expression system in CHO cells.

68.

Field R, Cockett M, Froud SJ (1988) Glutamine synthetase amplification of TIMP expression in CHO cells. In: Advances in Cell Biology and Technology for Bioprocesses, Butterworths, pp195-197. ♦ 80mg/L TIMP made in fermenters.

17.

Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Preneta A, Stephens PE, Thompson S, Yarranton GT (1989) The development of a process for the production of HIV1 GP120 from recombinant cell lines. In: Production of Biologicals from Animal Cells in Culture, Butterworths, pp110-116. ♦ GP120 expressed in amplified GS-CHO. Production levels of 1-3mg/l Cleavage

of GP120 coincides with depletion of asparagine, glutamate, aspartate and serine from medium.

19.

Gastinel LN, Simister NE, Bjorkman PH (1992) Expression and crystallisation of a soluble and functional form of an Fc receptor related to class 1 histocompatibility molecules. Proc. Natl. Acad. Sci. 89(2): 638-642. ♦ Expression of neonatal Fc receptor (cell surface and soluble forms) in amplified

GS-CHO. Soluble form gave ca. 40mg/l. Cells also grown in hollow fibre reactor.

20.

Gjorloff A, Hedlund G, Kallard T, Sarson D, Fischer H, Trowsdale J, Sjogren HO, Dohlsten M (1992) The LFA-3 adhesion pathway is differently utilised by super-antigen-activated human CD4+ T-cell subsets. Scand. J. Immunol. 36(2):243-250. ♦ HLA – DR4 and LFA3 expressed in amplified GS-CHO. Protein expressed on

cell membrane.

21.

Gloor S, Nasse K, Essen LO, Appel F (1992) Production and secretion in CHO 22.


cells of the extracellular domain of AMOG.beta 2, a type-11 membrane protein. Gene 120(2):307-312. ♦ Recombinant AMOG.beta 2 membrane protein secreted by amplified GS-CHO

cells and concentrations of 15mg/L achieved. Guerini D, Schroder S, Foletti D, Carafoli E (1995) Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane

Ca2+ ATPase. J. Biol. Chem. 270:14643-14650. ♦ GS – CHO cell lines created. Initial transfectants were subjected to additional

rounds of selections with MSX resulting in clones with 4 to 10 gene copies per cell. The stability of expression of transfected cells was examined in the presence and absence of selective drug (MSX). In the absence of MSX productivity was retained over short culture periods but was substantially decreased after 30 to 40 passages. In contrast stable production was maintained for 6 months in the presence of MSX.

67.

Gutman O, Danieli T, White JM, Henis YI (1993) Effects of exposure to low pH on the lateral mobility of influenza haemagglutinin expressed at the cell surface. Correlation between mobility inhibition and inactivation. Biochem. 32:101-106. ♦ HA expressed on surface of amplified GS-CHO cells.

25.

Harfst E , Johnstone AP (1992) Characterisation of the glutamine synthesase amplifiable eukaryotic expression system applied to an integral membrane protein – the human thyrotropin receptor. Anal. Biochem. 207:80-84. ♦ Thyrotropin expressed on surface of CHO cells using GS selection vector at

levels at least 10 x that achieved in other systems. Receptor is efficiently coupled to adenylate cyclase.

26.

Harfst E, Johnstone AP, Gout I, Taylor AH, Waterfield MD, Nussey SS (1992a) The use of the amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyrotrophin receptor. Mol. Cell. Endocrinol. 83(2-3):117-123.

♦ Functional Thyrotrophin receptor expressed at high level on surface of GS-CHO. In comparison Baculovirus system gave no demonstrable protein production.

27.

Harfst E, Johnstone AP, Nussey SS (1992b) Characterisation of the extracellular region of the human thyrotropin receptor expressed as a recombinant protein. J. Mol. Endocrinol. 9:227-236. ♦ Describes isolation and partial characterisation of recombinant thyrotropin

receptor from GS-CHO cells.

28.

Harfst E, Johnstone AP, Nussey SS (1992c) Interaction of thyrotropin and thyroid-stimulating antibodies with recombinanct extracellular region of human TSH receptor. Lancet 339:193 - 194. ♦ CHO cells expressing recombinant thyrotropin receptor from a GS-vector used

to study interaction of receptor with autoantibodies from sera of patients with autoimmune disease.

29.

Hovey A, Bebbington CR, Jenkins N (1994) Simultaneous control of growth and 70.


productivity using a mutant CHO cell line. In Animal Cell Technology: Products of today, prospects for tomorrow. eds R.E. Spier, J.B. Griffiths and W.Berthold. Butterworth–Heinemann, pp422-424. ♦ Heat shock induction used to produce recombinant TIMP from a mutant CHO

cell line. TIMP was expressed in a GS vector. Hovey A, Bebbington CR, Jenkins N (1994) Control of growth and recombinant protein synthesis by heat-shock in a mutant mammalian cell line. Biotechnol. Letters. 16:215-220. ♦ Describes use of heat shock to induce recombinant protein (TIMP) synthesis in

a temperature sensitive CHO cell line. TIMP expressed in a GS vector.

75.

Jenkins N , Hovey A (1993) Generation of CHO cell mutants for growth control. In Kaminogawa, S. et al. (eds) Animal Cell Technology: Basic and Applied Aspects 5, pp267-272. ♦ Created temperature sensitive mutant of CHO expressing TIMP from GS

vector. Temperature switching arrested cell growth and increased product yield.

76.

Jenkins N , Hovey A (1993) Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotech. Bioeng. 42:1029-1036. ♦ Temperature sensitive mutants of CHO cells were transfected with the gene for

TIMP using GS amplification. Alternating incubation between permissive (34oC)

and non permissive (39°C) temperatures arrested cell growth whilst maintaining high cell viability for up to 170 hours. TIMP production rate was increased 3 to 4

fold over the period of arrest compared with controls at 34° resulting in a 35% increase in yield.

69.

Kemble GW, Henis YI, White JM (1993) GPI- and transmembrane anchored influenza haemagglutinin differ in structure and receptor binding activity. J. Cell Biol. 122:1253-1265. ♦ Expressed haemagglutinin including engineered variants in amplified GS-CHO

cells.

31.

King DJ, Byron OD, Mountain A, Weir N, Harvey A, Lawson ADG, Proudfoot KA, Baldock D, Harding SE, Yarranton GT, Owens RJ (1993) Expression, purification and characterization of B72.3 Fv fragments. Biochem. J. 290:723-729. ♦ Fv expressed in GS-CHO and in E.coli. Yields of 4mg/L achieved in CHO roller

bottles and 450 mg/Lin E. coli fermentations.

32.

Kuwae S, Ohda T, Tamashima H, Miki H. Kobayashi K (2005) Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells. J. Biosci. Bioeng. 100:502-510. ♦ Expressed human antithrombin at 1 g/L using GS-CHO cells..

145.

Laubach VE, Garvey EP, Sherman PA (1996) High-level expression of human inducible nitric oxide synthase in Chinese hamster ovary cells and characterisation

88.


of the purified enzyme. Biochem. Biophys. Res. Comm. 218:802-807.

♦ iNOS expressed in CHO cells using GS system. Amplification in the presence of 400 µM MSX increased product levels 3 to 4 fold. Expression was enhanced by sodium butyrate (2 mM). Expression levels were at least 20-fold higher than those reported for a baculovirus expression system. Also noted that sodium butyrate could restore high level expression in some cell lines in which iNOS expression declined with time in culture.

McCall M N, Shotton DM, Barclay AN (1992) Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies. Immunol. 76:310-317. ♦ Four soluble isoforms of CD45 expressed in GS-CHO at 5mg/L of spent culture

medium.

35.

McKnight AJ , Classon BJ (1992) Biochemical and immunological properties of rat recombinant interleukin 2 and interleukin 4. Immunol. 75:286-292. ♦ IL-2 and IL-4 expressed in GS-CHO.

36.

Moore JP, McKeating JA, Jones IM, Stephens PE, Clements G, Thomson S, Weiss RA (1989) Characterisation of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4. AIDS 4:307-315. ♦ Compares properties of GP120 made in GS-CHO cells with that made in insect

cells. The CHO derived protein bound SCD4 with affinity similar to viral GP120. Product from insect cells had lower affinity.

38.

Quilliam AL, Osman N, McKenzie IFC, Hogarth PM (1993) Biochemical characterization of murine FcαRI. Immunol. 78:358-363. ♦ Fc receptor expressed on surface of GS-CHO cells.

44.

Sano H, Chiba H, Iwaki D, Sohma H, Voelker DR, Kuroki Y (2000) Surfactant proteins A and D bind CD14 by different mechanisms. J. Biol. Chem. 275:22442 -22451. ♦ Used GS expression system with CHOK1 and pEE14.4 to express CD14 from a

generated stable cell line. An insect cell line was also used for comparison. Mammalian expressed CD14 consistent with insect expression. Lung surfactant proteins SP-A and D are important in the innate immunity of the lung and were investigated in to their mechanism for binding CD14.

120.

Springer S, Döring K, Skipper JCA, Townsend ARM, Cerundolo V (1998) Fast association rates suggest a conformational change in the MHC class 1 molecule H-2Db upon peptide binding. Biochem. 37:3001-3012. ♦ Soluble H-2Db class 1 molecules expressed in CHO cells using GS systems.

Several rounds of amplification used.

89.

Trowbridge IS, Johnson P, Ostergaard H, Hole N (1992) Structure and function of CD45: a leukocyte-specific protein tyrosine phosphatase. Adv. Exp. Med. Biol. 323:29-37. ♦ CD45 external domain expressed at 20 mg/L in GS CHO cells.

48.


Williams AF, Davis SJ, He Q, Barclay AN (1989). Structural diversity in domains of the immunoglobulin superfamily. Cold Spring Harb. Symp. Quart. Biol. 54:Pt 2, 637-647. mead ♦ Expression of soluble rat CD4 in amplified GS-CHO.

49.

SECTION 6: Product Characteristics and Critical Quality Attributes

Baker KN, Rendal, MH, Hills AE, Hoare M, Freedman RB, James DC (2001) Metabolic control of recombinant protein N – glycan processing in NS0 and CHO cells. Biotech. Bioeng. 73:188-202. ♦ The authors compare glycosylation of TIMP (tissue inhibitor of

metalloproteinases 1) made in GS-NS0 and GS-CHO. Significant differences were found. In NS0 derived material, 30% of N-glycan antennae terminated in alpha 1, 3 linked galactose (none found in CHO). The level of sialylation was slightly higher in NS0 than CHO. Sialic acid from CHO was predominantly N-acetylneuraminic acid whilst NS0 – derived glycan contained equal proportions of N-glycolyl and N-acetyl variants. It was demonstrated that manipulation of nucleotide - sugar metabolism (by addition of precursors) could be used to modify glycan processing.

104.

Bebbington CR (1995) Expression of antibody genes in mammalian cells. In Zola, H. (ed). "Monoclonal antibodies: the next generation" Bios Scientific. (Oxford) pp165-181. ♦ Review of expression systems including GS. Also discusses glycosylation of

antibodies.

71.

Chen P, Cataldo S, Dennis P, Popoloski J, Dabora R (1994) A comparison of Chinese hamster ovary (CHO) and mouse myeloma (NS0) cell lines producing a recombinant protein . American Chem. Soc. Abs. of 207th National Meeting San Diego. ♦ Rec protein expressed at level of 120 mg/L with DHFR-CHO in suspension and

at 450 mg/L in GS-NS0 in batch culture (following medium optimisation). Substantial differences seen in the carbohydrate structure and pharmacokinetic properties of the protein produced in the two cell lines.

11.

Dorai H , Nemeth JF, Cammaart E Wang Y, Tang QM, Magill A, Lewis MJ, Raju TS, Picha K, O'Neil K, Ganguly S, Moore G. (2009) Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODYTM: Factors that influence productivity and product quality. Biotechnol. Bioeng. 103:162-176.

173.

Flesher AR, Marzowski J, Wang W-C, Raff HV (1995) Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion protein: correlations of oligosaccharide content with in-vivo clearance profile. Biotech. Bioeng. 46:399-407.

♦ Compared glycosylation and in vivo clearance of a CTLA4/immunoglobulin

fusion protein made in GS-NS0 cells with that made in CHO cells. NS0 derived

66.


protein had no detectable N-acetylneuraminic acid and had concomitant accelerated clearance.

Fu J, Bongers J, Tao L, Huang D, Ludwig R, Huang Y, Qian Y, Basch , Goldstein J, Krishnan R, You L, Li ZJ, Russell RJ. (2012) Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines. J. Chromat. B 908:1-8.

216.

Gramer MJ , Goochee CF (1994) Glycosidase activities of the 293 and NS0 cell lines, and of an antibody producing hybridoma cell line. Biotechnol. Bioeng. 43:423-428. ♦ Several glycosidases detected in NS0 cell lysates. Sialidase present and stable

at pH 4.5 but unstable at 7.5. CHO sialidase stable at 7.5.

24.

Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, Priem P, van den Bremer ETJ, Gerritsen J, van Berkel PHC (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol. Bioeng. 108:1591-1602.

200.

Hansen R, Dickson AJ, Goodacre R, Stephens GM, Sellick CA (2010) Rapid characterization of N-linked glycans from secreted and gel-purified monoclonal antibodies using MALDI-ToF mass spectrometry. Biotechnol. Bioeng. 107:902-908.

195.

Hills AE , Patel A, Boyd P, James DC (2001) Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnol. Bioeng. 75:239-251. ♦ Describes impact of manipulating intracellular pools of UDP-sugars upon

glycosylation of an IgG4.

144.

Kilgore BR , Lucka A, Patel R, Andrien B, Dhume ST (2005) Cell line Switch from NS0 to CHO Causes Several Changes in Glycosylation of Recombinant IgG. ACS 33rd Northeast Regional Meeting (Abstract) ♦ http://acs.confex.com/acs/nerm05/techprogram/P21476.HTM

214.

Lifely MR, Hale C, Boyce S, Keen MJ, Phillips J (1995) Glycosylation and biological activity of Campath - IH expressed in different cell lines and grown under different culture conditions. Glycobiol. 5:813-822. ♦ Antibody expressed in CHO (DHFR) rat YO myeloma and NS0 (GS) cells.

Glycosylation varied significantly between cell lines and to a minor extent depending on culture conditions. YO antibody was unusual in having a bisecting GlcNAc on the core oligoscaccharide and NS0 antibody appeared to be under glycosylated. YO antibody had enhanced activitity in ADCC assays compared with CHO or NS0.

74.

Reid CQ, Tait AS, Baldascini H, Mohindra A, Racher AJ, Bilsborough S, Smales CM, Hoare M (2010) Rapid whole monoclonal antibody analysis by mass spectrometry: An ultra scale-down study of the effect of harvesting by centrifugation on the post-translational modification profile. Biotechnol. Bioeng. 107:85-95.

188.

Robinson DK, Chan CP, Yu Ip, CC, Seamans TC, Lee DK, Lenny AB, Tung J-S, DiStefano DJ, Munshi S, Gould SL, Tsai PK, Irwin J, Mark GE, Silberklang M (1994) Product consistency during long-term fed-batch culture. In:Animal Cell

46.


Technology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, Griffiths JB, Berthold W, Butterworths-Heinemann, pp763-767. ♦ Expressing antibody genes in GS-NS0. Yields up to 1.8g/L in three to four week

duration fed batch. Glycoform pattern shifts towards incomplete mannose-terminated glycans as culture progresses.

Robinson DK, Chan CP, Yu Ip, C, Tsai PK, Tung J, Seamans TC, Lenny AB, Lee DK, Irwin J, Silberklang M (1994) Characterization of a recombinant antibody produced in the course of a high yield fed-batch process. Biotech. Bioeng. 44:727-735. ♦ Anti-HIV gp120 Mab expressed in non-amplified GS-NS0 system. Fed-batch

process developed which increased yields ten-fold compared with batch culture. The clone produced 0.86g mAb/L over 22 days. Antigen binding and MWt remained constant throughout culture. Four major IEF bands were observed with a minor fifth more acidic band appearing later in culture. Pattern of glycoforms secreted by cells changes as culture progresses.

52.

Rossman C, Sharp N, Allen G, Gewert D (1996) Expression and purification of recombinant, glycosylated human interferon alpha 2b in murine myeloma NS0 cells. Protein Expression and Purification 7:335 – 342. ♦ Expressed Interferon Alpha in GS-NS0 cells and isolated a cell line expressing

20 µg/106 cells/24h and accumulating 120 µg/mLl in culture. Glycosylation was similar to that of non recombinant interferon purified from human Namalwa cells. This is the highest reported level of glycosylated, recombinant IFN expression in a stable mammalian system.

85.

van Berkel PHC , Gerritsen J, Perdok G, Valbjørn J, Vink T, van de Winkel JGJ, Parren PWHI. (2009) N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG. Biotechnol. Prog. 25:244-251.

174.

Yu Ip CC, Miller WJ, Silberklang M, Mark GE, Ellis RW, Huang L, Glushka J, Van Halbeek H, Zhu J, Alhadeff JA (1994) Structural characterisation of the N-Glycans of a humanised Anti-CD18 murine immunoglobulin G. Arch Biochem. Biophys. 308:87 – 399. ♦ Identifies glycan structures of humanised IgG4 expressed in GS-NS0.

51.

SECTION 7: Culture Conditions for GS Cell Lines including Media and Feeding Strategies

Bibila TA, Ranucci CS, Glazomitsky K, Buckland BC, Aunins JG (1994) Monoclonal antibody process development using medium concentrates. Biotechnol. Prog. 10:87-96. ♦ Describes fed-batch process using concentrated medium with GS-NS0 cell lines

making recombinant antibodies. Up to 7-fold increases in antibody titre were achieved compared with batch culture.

54.

Bibila TA , Robinson DK (1995) In pursuit of the optimal fed-batch process for monoclonal antibody production. Biotechnol. Prog. 11:1-13.

60.


♦ A review of approaches taken to optimise fed-batch processes for antibody production.

Birch JR, Boraston RC, Metcalfe H, Bebbington CR, Field RP (1994) Selecting and designing cell lines for improved physiological characteristics. Cytotechnol. 15:11-16. ♦ Describes transfection of GS into hybridoma to give glutamine independence.

Productivity was increased in glutamine free medium. Also describes the isolation of a cholesterol independent variant of the NS0 myeloma cell line.

58.

Birch JR , Froud S (1994) Mammalian cell culture systems for recombinant protein production. Biologicals 22:127-133. ♦ Gives example of MAb production (200 mg/L) using GS-NS0 cells in protein-free

medium.

53.

Broad D, Boraston R, Rhodes M (1991) Production of recombinant proteins in serum-free media. Cytotechnol. 5:47-55. ♦ Compares productivity of three amplified GS-CHO lines making recombinant

proteins in the presence and absence of serum (no significant differences seen).

8.

Brown ME, Renner G, Field RP, Hassell T (1992) Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system. Cytotechnol. 9:231-236. ♦ Describes improvements in batch process leading to antibody yields ranging up

to 1g/L. Serum-free medium used.

9.

Buser CW, Beaudet R, Soohoo N, Pugh GG (1994) Development of serum-free media for engineered NS0 cell lines. In Animal Cell Technology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, Griffiths JB, Berthold W. Butterworth-Heinemann, pp121-139. ♦ Describes development of serum-free medium for a GS-NS0 cell line, based on

Williams medium E. Lipid supplements added to the medium.

10.

Cannon -Carlson S, Varnerin J, Tsarbopoulos A, Jenh C-H, Cox MA, Chou C-C, Connelly N, Zavody P, Tang J C-T (1998) Expression purification and characterisation of recombinant human Interleukin-13 from NS0 Cells. Protein Expression and Purification 12:239-248. ♦ IL-13 expressed in NS0 cells using GS expression system. Cells grown in 15

litre fed batch culture. Details given of the medium and feed used. The protein is unglycosylated.

92.

Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. BioTechnol. 8:662-667. ♦ Yields of 180 mg/L achieved in shake flask culture. Sodium butyrate enhanced

production.

13.

Davis SJ, Ward HA, Puklaver MJ, Willis AC, Williams AF, Barclay AN (1990) High 15.


level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants. J. Biol. Chem. 265:10410-10418. ♦ S CD4 and S CD4 (half) expressed at levels of 80 and 25 µg/L from GS-CHO

cells. Used GMEM-S + 5% FCS and MSX (15 or 100µM) in roller bottles. Sodium butyrate added to enhance production. Also used switch to serum free medium (GMEM-S + 2 mM butyrate) at final stage, to aid purification.

DiStefano DJ, Mark GE, Robinson DK (1996) Feeding of nutrients delays apoptotic death in fed-batch cultures of recombinant NS0 myeloma cells. Biotechnol. Letters 18:1067 – 1072. ♦ Nutrient solutions added to fed-batch cultures of recombinant GS-NS0 cells

delayed onset of apoptosis and decreased rate of apoptotic death. This seems to be responsible for increase in culture longevity and five to ten fold increase in product concentration.

82.

Duncan PJ, Jenkins HA, Hobbs G (1997) The effect of hyperosmotic conditions on growth and recombinant protein expression by NS0 myeloma cells in culture. The Genetic Engineer and Biotechnologist 17:75-78. ♦ Hyperosmotic conditions (400 mOsm) led to a 1.8 fold increase in specific

productivity compared with control conditions (275 mOsm). The conditions also led to large increases in glucose and glutamate uptake rates and in lactate production rates.

93.

Fries S, Glazomitsky K, Woods A, Forrest G, Hsu A, Olewinski R, Robinson DK, Chartrain M (2005) Evaluation of disposable bioreactors. Rapid production of recombinant proteins by several animal cells. BioProcess International 3( Supp 6):36-44. ♦ Describes growth of antibody-producing GS-NS0 cell line in cell culture-bag

disposable bioreactor system.

155.

Frahm B , Lane P, Märkl H, Pörtner R (2003) Improvement of a mammalian cell culture process by adaptive, model-based dialysis fed-batch cultivation and suppression of apoptosis. Bioprocess Biosyst. Eng. 26:1-10.

149.

Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Preneta A, Stephens PE, Thompson S, Yarranton GT (1989) The development of a process for the production of HIV1 GP120 from recombinant cell lines. In:Production of Biologicals from Animal Cells in Culture, Butterworths, pp110-116. ♦ GP120 expressed in amplified GS-CHO. Production levels of 1-3 mg/L

Cleavage of GP120 coincides with depletion of asparagine, glutamate, aspartate and serine from medium.

19.

Hermes PA , Castro CD (2010) A fully defined, fed-batch, recombinant NS0 culture process for monoclonal antibody production. Biotechnol. Prog. 26:1411-1416.

193.

Hogwood CEM , Tait AS, Koloteva-Levine N, Bracewell DG, Smales CM. (2013) The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process. Biotechnol. Bioeng. 110:240-251.

219.


Jiang Z , Droms KA, Geng Z, Casnocha SA, Xiao Z, Gorfien SF, Jacobia SJ (2012) Fed-batch cell culture process optimisation. BioProcess Intl. 10:40-45

215.

Kadarusman J , Bhatia R, McLaughlin J, Lin WR (2005) Growing cholesterol-dependent NS0 myeloma cell line in the Wave bioreactor system: overcoming cholesterol-polymer interaction by using pretreated polymer or inert fluorinated ethylene propylene. Biotechnol. Prog. 21:1341-1346. ♦ Discusses choice of material for manufacture of disposable bioreactor systems

168.

Keen MJ, Hale C (1996) The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker. Cytotechnol. 18:207-217. ♦ Describes protein free medium for GS-NS0 supplemented with cholesterol,

phosphatidylcholine and β-cyclodextrin. Adapted cells to become cholesterol independent.

80.

Kuwae S, Ohda T, Tamashima H, Miki H, Kobayashi K (2005) Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells. J. Biosci. Bioeng. 100:502-510. ♦ Expressed human antithrombin at 1 g/L using GS-CHO cells. Developed fed-

batch process and evaluated the process at different pH values.

145.

Legmann R , Schreyer HB, Combs RG, McCormick EL, Russo AP, Rodgers ST (2009) A predictive high-throughput scale-down model of monoclonal antibody production in CHO cells. Biotechnol. Bioeng. 104:1107-1120.

185.

Ma N, Ellet J, Okediadi C, Hermes P, McCormick E, Casnocha SA (2009) A single nutrient feed supports both chemically defined NS0 and CHO fed-batch processes: Improved productivity and lactate metabolism. Biotechnol. Prog. 25:1353-1363.

182.

Metcalfe H, Field RP, Froud SJ (1994) The use of 2-hydroxy-2, 4, 6-cycloheptarin-1-one (tropolone) as a replacement for transferrin. In:Animal Cell Technology: Products of Today, Prospects for Tomorrow. Eds: Spier RG, Griffiths JB, Moignier B. Butterworth-Heinemann. pp 88-90. ♦ Tropolone used as a transferrin replacement for culture of GS-NS0 cells.

37.

Naciri M, Kuystermans D, Al-Rubeai M (2008) Monitoring pH and dissolved oxygen in mammalian cell culture using optical sensors. Cytotechnol. 57:245-250.

170.

Osman JJ, Birch JR, Varley J (2001) The response of GS-NS0 myeloma cells to pH shifts and pH perturbations. Biotechnol. Bioeng. 75:63-73. ♦ Describe the effects of pH shifts and perturbations on growth, productivity and

glucose metabolism for a GS-NS0 cell line.

108.

Osman JJ, Birch JR, Varley J (2002) The response of GS-NS0 myeloma cells to single and multiple pH perturbations. Biotech. Bioeng. 79:398–407. ♦ Used GS-NS0 mouse myeloma cells expressing cB72.3 antibody in stirred tank

batch fermentations. Analysed the effects of single and multiple pH perturbations at pH 8 and 9 from pH7.3. For multiple pH perturbations a 2

119.


component system was used. Increasing the number of perturbations or exposure time to changed pH increases cell death.

Ray NG, Rivera R, Gupta R, Mueller D (1997) Large scale production of humanised monoclonal antibody expressed in a GS-NS0 cell line. In: Animal Cell Technology, Ed: Carrondo MJT. Kluwer Academic Publishers pp235-241. ♦ Describes production of a humanised antibody from GS-NS0 cell line in a 2000

litre stirred tank reactor run in fed-batch mode. Achieved 370-470 mg antibody/L after approx. 10 days. Deals with scale-up issues. Demonstrates effect of sparge rate on lactate accumulation which is attributed to levels of carbon dioxide in the culture. Accumulation of CO2 at low sparge rates led to increased lactate accumulation.

81.

Robinson DK, Seamans TC, Gould SL, DiStefano DJ, Chan CP, Lee DK, Bibila T, Glazomitsky K, Munshi S, Daugherty B, O'Neill Palladino L., Stafford-Hollis J, Hollis GF, Silberklang M (1994) Optimization of a fed-batch process for production of a recombinant antibody. Reprinted from Biochemical Engineering VIII, Ann. N.Y. Acad. Sci. 745:285-296. ♦ Describes the development of an optimized fed-batch process for a GS-NS0 cell

line secreting a monoclonal antibody. Includes data on amino acid consumption rates.

62.

Robinson DK, DiStefano D, Gould SL, Cuca G, Seamans TC, Benincasa D, Munshi S, Chan CP, Stafford-Hollis J, Hollis GF, Jain D, Ramasubramanyan K, Mark GE, Silberklang M (1995) Production of engineered antibodies in myeloma and hybridoma cells. In: Antibody Engineering. Eds: Wang H, Imanaka T. Worthington: ACS, pp1-14. ♦ Detailed description of production of recombinant antibody in a GS-NS0 system.

84.

Sanders PG , Wilson RH (1984) Amplification and cloning of the Chinese hamster glutamine synthetase gene. The EMBO Journal 3:(1), 65-71. ♦ Descibes amplification of endogenous GS gene in CHO using MSX selection.

Describes modified GMEM with glutamate, asparagine, nucleotides and pyruvate.

47.

Seamans TC, Gould SL, DiStefano DJ, Silberklang M, Robinson DK (1994) Use of lipid emulsions as nutritional supplements in mammalian cell culture. Annals N.Y. Acad. Sci. 745:240-243. ♦ Describes preparation of stable lipid emulsions to substitute for lipoproteins in

the culture of NS0 cells secreting a recombinant antibody.

61.

Seamans TC, Fries S, Beck A, Wurch T., Chenu S, Chan C, Ushio M, Bailey J, Kejariwal A, Ranucci C, Villani N, Ozuna S, Goetsch L, Corvaia N, Salmon P, Robinson DK, Chartrain M (2008) Cell cultivation process transfer and scale-up in support of production of early clinical supplies of an anti IGF-1R antibody, Part 1. BioProcess International 6(3):26-36.

163.

Seamans TC, Fries S, Beck A, Wurch T., Chenu S, Chan C, Ushio M, Bailey J, Kejariwal A, Ranucci C, Villani N, Ozuna S, Goetsch L, Corvaia N, Salmon P, Robinson DK, Chartrain M (2008) Cell cultivation pocess transfer and scale-up in support of production of early clinical supplies of an anti IGF-1R antibody, Part 2.

164.


BioProcess International 6(4):34-42. Sellick CA, Hansen R, Jarvis RM, Maqsood AR, Stephens GM, Dickson AJ, Goodacre R (2010) Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics. Biotechnol. Bioeng. 106:432-442.

187.


212.

Silk NJ, Denby S, Lewis G, Kuiper M, Hatton D, Field RP, Bagnaz F, Lye GJ (2010) Fed-batch operation of an industrial cell culture process in shaken microwells. Biotechnol. Lett. 32:73-78.

♦ Describes a fed-batch culture of a GS-CHO cell line in a shaken 24-well plate.

183.

Titchener -Hooker NJ , Dunnill P, Hoare M (2008) Micro biochemical engineering to accelerate the design of industrial-scale downstream processes for biopharmaceutical proteins. Biotechnol. Bioeng. 100:473-487.

167.

Velez-Suberbie ML, Tarrant RDR, Tait AS, Spencer DIR, Bracewell DG. (2013) Impact of aeration strategy on CHO cell performance during antibody production. Biotechnol. Prog. 29:116-126.

220.

Wayte J, Boraston R, Bland H, Varley J, Brown M (1997) pH: effects on growth and productivity of cell lines producing monoclonal antibodies: control in large-scale fermenters. The Genetic Engineer and Biotechnol. 17:125-132.

♦ Describes effect of pH on growth and productivity of a GS-NS0 cell line making a humanised monoclonal antibody. Productivity increased at pH7.1 compared with 7.4.

94.

Zhang J, Robinson DK (2005) Development of animal-free, protein-free and chemically-defined media for NS0 culture. Cytotechnol. 48:59-74.

156.

Zhou W, Bibila T, Glazomitsky K, Montalvo J, Chan C, DiStefano D, Munshi S, Robinson D, Buckland B, Aunins J (1996) Large-scale production of recombinant mouse and rat growth hormone by fed-batch GS-NS0 cell cultures. Cytotechnol. 22:239-250. ♦ Transfected NS0 cells with GS vector containing growth hormone genes and

sequence for targeting integration by homologous recombination (for optimal expression). Operated fed-batch cultures at 25 litrel scale and information on operating conditions is given. Yields of mouse and rat growth hormone were 580 and 240 mg/L respectively. Data presented on cell metabolism. Glucose consumption rate decreased during transition to stationary phase and lactate was consumed during stationary phase.

77.

Wu MH, Dimopoulos G, Mantalaris A, Varley J (2004) The effect of hyperosmotic pressure on antibody production and gene expression in the GS-NS0 cell line. Biotechnol. Appl. Biochem. 40:41-46. ♦ Describes growth and productivity kinetics as well as gene expression, using

microarray technology, at various osmolalities..

128.


Zhou W, Chen C-C, Buckland B, Aunins J (1997) Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production. Biotech. Bioeng. 55:783-792. ♦ Describes feeding strategy leading to final antibody concentration in excess of

2.7g/L. Describes transitions in metabolism caused by nutrient depletion. The cell line used in the study had three vector copies.

90.

SECTION 8: Metabolism of GS Cell Lines

Bell SL, Bebbington CR, Bushell ME, Sanders PG, Scott MF, Spier RE, Wardell JN (1991) Genetic engineering of cellular physiology. In: Production of Biologicals from Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp 304-306. ♦ GS gene used to confer glutamine independence on a hybridoma.

4.

Bell SL, Bushell ME, Scott MF, Wardell JN, Spier RE, Sanders PG (1992) Genetic modification of hybridoma glutamine metabolism: physiological consequences. In Animal Cell technology: Developments, Processes and Products. Eds: Spier RE, Griffiths JB, MacDonald C. Butterworth-Heinemann, pp180-182. ♦ GS gene transfected into hybridoma cell to confer glutamine independence.

5.

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushell ME Sanders PG (1995) Genetic engineering of hybridoma glutamine metabolism. Enzyme and microbial technol. 17:98-106. ♦ GS gene used to confer glutamine independence on a hybridoma.

65.

Bibila TA , Ranucci C, Glazomitsky K, Buckland BC, Aunins JG (1994) Investigation of NS0 cell metabolic behaviour in monoclonal antibody producing clones. Ann. N.Y. Acad. Sci. 745:277-284.

146.

Birch JR, Boraston RC, Metcalfe H, Bebbington CR, Field RP (1994) Selecting and designing cell lines for improved physiological characteristics. Cytotechnol. 15:11-16. ♦ Describes transfection of GS into hybridoma to give glutamine independence.

Productivity was increased in glutamine free medium.

58.

Bond J , Varley J (2005) Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells. Cyometry Part A 64A:43-50. ♦ Evaluates methods for measurement of intracellular pH.

125.

Cairo JJ, Paredes C, Godia F, Prats E, Azorin F, Cornudella L (1998) Modification of hybridoma cells metabolism. In: New Developments and New Applications in Animal Cell Technology. Eds: Merten O-W, Perrin P, Griffiths B. Kluwer Academic Publishers, pp167-174.

♦ Transfection of hybridoma with GS gene eliminated glutamine requirement and suppressed ammonia production. Also resulted in halving of glucose uptake rate whilst maintaining similar growth pattern. Discusses metabolic fluxes in cells with and without GS.

86.


Dempsey J, Ruddock S, Osborne M, Ridley A, Sturt S, Field R (2003) Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase. Biotechnol. Prog. 19:175-178.

♦ Repeated nutrient analysis and re-supplementation of serum-free antibody producing NS0 cultures performed. As a result media and feeds were developed which gave 10-fold increases in antibody harvest titres up to 600 mg/L.

109.

DiStefano, DJ, Mark GE, Robinson DK (1996) Feeding of nutrients delays apoptotic death in fed-batch cultures of recombinant NS0 myeloma cells. Biotechnol. Letters 18:1066 – 1073. ♦ Nutrient solutions added to fed-batch cultures of recombinant GS-NS0 cells

delayed onset of apoptosis and decreased rate of apoptotic death. This seems to be responsible for increase in culture longevity and five- to- ten- fold increase in product concentration.

82.

Dorai H. Kyung YS, Ellis D, Kinney C, Lin C, Jan D, Moore G, Betenbaugh MJ (2009) Expression of anti-apoptosis genes alters lactate metabolism of Chinese hamster ovary cells in culture. Biotechnol. Bioeng. 103:592-608.

177.

Downham MR, Farrell WE, Jenkins HA (1996) Endoplasmic reticulum protein expression in recombinant NS0 Myelomas grown in batch culture. Biotech. Bioeng. 51:691 – 696. ♦ Production of ER proteins (GRP78/BiP, GRP94 and Erp72) increased during

decline phase of batch culture of GS-NS0 coincident with an increase in production rate of recombinant antibody and reduction in uptake of glucose and glutamate.

83.

Duncan PJ, Jenkins HA, Hobbs G (1997) The effect of hyperosmotic conditions on growth and recombinant protein expression by NS0 myeloma cells in culture. The Genetic Engineer and Biotechnol. 17:75-78. ♦ Hyperosmotic conditions (400 mOsm) led to a 1.8 fold increase in specific

productivity compared with control conditions (275 mOsm). The conditions also led to large increases in glucose and glutamate uptake rates and in lactate production rates.

93.



111.

Kearns B, Lindsay D, Manahan M, McDowall J, Rendeiro D (2003) NS0 batch cell culture process characterisation: a case study. BioProcessing Journal 2:52-57. ♦ Investigation into decline in productivity as cell culture generation number

increased. No genetic instability seen but a phenotypic instability, related to the metabolic state of the culture, is reported.

110.


Khoo SHG, Al-Rubeai M (2009) Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line. Metabol. Eng. 11:199-211.

178.

Ma N, Ellet J, Okediadi C, Hermes P, McCormick E, Casnocha SA (2009) A single nutrient feed supports both chemically defined NS0 and CHO fed-batch processes: Improved productivity and lactate metabolism. Biotechnol. Prog. 25:1353-1363.

182.

McKenna SL , Cotter TG (2000) Inhibition of caspase activity delays apoptosis in a transfected NS0 myeloma cell line. Biotech. Bioeng. 67:165-176. ♦ Z-VAD-fmk, a specific inhibitor of caspases reduced apoptosis in GS-NS0 cells

but did not increase productivity. The inhibitor did not prevent mitochondrial dysfunction.

102.

Paredes C, Prats E, Cairo JJ, Azoris F, Cornudella Ll, Godia F (1999) Modification of glucose and glutamine metabolism in hybridoma cells through metabolic engineering. Cytotechnol. 30:85-93. ♦ Transfected a hybridoma cell line with GS. Transfected cells had a reduced

growth rate and a lower glucose utilisation rate. Ammonia and alanine production was eliminated. Alanine was required for optimal growth.

101.

Ray NG, Rivera R, Gupta R, Mueller D (1997) Large scale production of humanised monoclonal antibody expressed in a GS-NS0 cell line. In: Animal Cell Technology. Ed: Carrondo MJT Kluwer Academic Publishers pp235-241. ♦ Describes production of a humanised antibody from GS-NS0 cell line in a 2000l

stirred tank reactor run in fed-batch mode. Achieved 370-470mg/antibody/l after approx. 10 days. Deals with scale-up issues. Demonstrates effect of sparge rate on lactate accumulation which is attributed to levels of carbon dioxide in the culture. Accumulation of CO2 at low sparge rates led to increased lactate accumulation.

81.

Sellick CA, Hansen R, Maqsood AR, Dunn WB, Stephens GM, Goodacre R, Dickson AJ (2009) Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells. Anal. Chem. 81:174-183.

196.

Sellick CA, Hansen R, Jarvis RM, Maqsood AR, Stephens GM, Dickson AJ, Goodacre R (2010) Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics. Biotechnol. Bioeng. 106:432-442.

187.


212.

Sengupta N, Rose ST, Morgan JA (2011) Metabolic flux analysis of CHO cell metabolism in the late non-growth phase. Biotechnol. Bioeng. 108:82-92.

202.

Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000) Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotech. Bioeng. 68:31-43. ♦ Transfection with the control expression vector (Neo marker) used in this study

99.


and exposure to the selective drug G418 led to upregulation of endogenous Bcl-2. This led to an increase in cell viability in cell cultures and prolonged survival. There was no influence on antibody titre.

Veraitch FS, Al-Rubeai M (2005) Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis. Biotechnol. Bioeng. 92:589-599.

123.

Zhou W, Bibila T, Glazomitsky K, Montalvo J, Chan C, Di Stefano D, Munshi S Robinson D, Buckland B, Aunins J (1996) Large-scale production of recombinant mouse and rat growth hormone by fed-batch GS-NS0 cell cultures. Cytotechnol. 22:239-250. ♦ Transfected NS0 cells with GS vector containing growth hormone genes and

sequence for targeting integration by homologous recombination (for optimal expression). Operated fed-batch cultures at 250 litre scale and information on operating conditions is given. Yields of mouse and rat growth hormone were 580 and 240 mg/L respectively. Data presented on cell metabolism. Glucose consumption rate decreased during transition to stationary phase and lactate was consumed during stationary phase.

77.

SECTION 9: Virology of NS0 Cell Lines

Froud SJ , Birch JR, McLean C, Shepherd AJ, Smith KT (1997) Viral contaminants found in mouse cell lines used in the production of biological products. In: Animal Cell Technology: From Vaccines to Genetic Medicine (Eds: Carrondo MJT, Griffiths JB, Moreira JL), Kluwer Academic Publishers, Dordrecht, pp681-686. ♦ Authors present data showing that, whilst it may not be possible to detect

infectious retrovirus in final bulk harvest of GS-NS0 fermentations, it may have been present at high titre up to this point..

139.

Taylor FR, Ferrant JL, Foley SF, Zeng C, Sernatinger J, Juffras R, Pepinsky B (2000) Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line. J. Biotech. 84:33-43. ♦ Large numbers of retroviral particles were observed in an NS0 subclone using

EM. Titres by infectivity assay were low and had the host range expected for a murine amphotropic retrovirus. Analysis of viral Gag Proteins indicated the presence of at least two closely related viruses N-terminal sequencing indicates that the viruses belong to the murine leukemia retrovirus family. A western blot assay using an antibody for the capsid protein was developed and may be useful for monitoring viral titre and clearance.

103.

SECTION 10: ‘Omic Studies of GS Cell Lines and Systems Biology

Ahmad N, Zhang J, Brown PJ, James DC, Birch JR, Racher AJ, Smales CM (2006) On the statistical analysis of the GS-NS0 cell proteome: imputation, clustering and variability testing. Biochem. Biophys. Acta 1764:1169-1187.

132.

Alete DE, Racher AJ, Birch JR, James DC, Smales CM (2005) The functional competence of animal cells: analysis of the secretory pathway. In: Animal Cell Technology meets Genomics (Eds: Gòdia F, Fussenegger M) Springer, Dordrecht, pp71-74.

129.


Alete DE, Racher AJ, Birch JR, Stansfield SH, James DC, Smales CM (2005) Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities. Proteomics 5:4689-4704.

122.

Charaniya S, Karypis G, Hu W-S (2009) Mining transcriptome data for function-trait relationship of hyper productivity of recombinant antibody. Biotechnol, Bioeng. 102:1654-1669.

172.

Dinnis DM, Stansfield SH, Schlatter S, Smales CM, Alete D, Birch JR, Racher AJ, Marshall CT, Nielsen LK, James DC (2006) Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol Bioeng 94:830-841.

133.

Khoo SHG, Falciani F, Al-Rubeai M (2007) A genome-wide transcriptional analysis of producer and non-producer NS0 myeloma cell lines. Biotechnol. Appl. Biochem. 47:85-95.

152.

Khoo SHG, Al-Rubeai M (2009) Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line. Metabol. Eng. 11:199-211.

178.

Krampe B , Swiderek H, Al-Rubeai M (2008) Transcriptome and proteome analysis of antibody-producing mouse myeloma NS0 cells cultivated at different cell densities in perfusion culture. Biotechnol. Appl. Biochem. 50:143-151.

165.

Krampe B , Fagan A, Gaora PÓ, Al-Rubeai M (2011) Chemostat-based transcriptional analysis of growth rate change and BCL-2 over-expression in NS0 cells. Biotechnol. Bioeng. 108:1603-1615. ♦ The GS-NS0 cell line described by Al-Rubeai and co-workers as ‘6A1’ is in fact

the cell line 6A1(100)3 (Bebbington et al 1992, reference #3).

199.

Mead EJ, Chiverton LM, Spurgeon SK, Martin EB, Montague GA, Smales CM, von der Haar T. (2012) Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines. PLOS One 7(10):e47422. ♦ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468484/

221.

McLeod J , O'Callaghan PM, Pybus LP, Wilkinson SJ, Root T, Racher AJ, James DC (2011) An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer. Biotechnol. Bioeng. 108:2193-2204.

197.

Sellick CA, Hansen R, Jarvis RM, Maqsood AR, Stephens GM, Dickson AJ, Goodacre R (2010) Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics. Biotechnol. Bioeng. 106:432-442.

187.

Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ, Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol. Bioeng. 88:474 – 488. ♦ Proteomic analysis of four different NS0 cell lines, with different levels of

118.


antibody production, identified a number of proteins with altered abundances. Amongst these proteins a number of molecular chaperones involved in folding and assembly of immunoglobulins were identified. The authors also report an abundance of light chain protein over heavy chain protein a likely prerequisite for efficient MAb production.

Smales CM, Birch JR, Racher AJ, Marshall CT, James DC (2003) Evaluation of individual protein errors in silver-stained two-dimensional gels. Biochem. Biophys. Res. Comm. 306:1050-1055.

136.

Stansfield SH , Allen EE, Dinnis DM, Racher AJ Birch JR James DC (2007) Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture. Biotechnol. Bioeng. 97:410-424.

151.

Swiderek H , Al-Rubeai M (2007) Functional genome-wide analysis of antibody producing NS0 cell line cultivated at different temperatures. Biotechnol. Bioeng. 98:616-630. ♦ Erratum: Biotechnol. Bioeng. (2008) 100:838

153.

Swiderek H, Logan A, Al-Rubeai M (2008) Cellular and transcriptomic analysis of NS0 cell response during exposure to hypoxia. J. Biotechnol. 234:103-111.

166.

Wu MH, Dimopoulos G, Mantalaris A, Varley J (2004) The effect of hyperosmotic pressure on antibody production and gene expression in the GS-NS0 cell line. Biotechnol. Appl. Biochem. 40:41-46. ♦ Describes growth and productivity kinetics as well as gene expression, using

microarray technology, at various osmolalities.

128.

SECTION 11: Cell Line Engineering

Astley K , Naciri M, Racher AJ, Al-Rubeai M. (2007) The role of p21cip1 in adaptation of CHO cells to suspension and protein-free culture. J Biotechnol 130:282-290

158.

Astley K, Al-Rubeai M (2008) The role of Bcl-2 and its combined effect with p21CIP1 in adaptation of CHO cells to suspension and protein-free culture. Appl. Microbiol. Biotechnol. 78:391-399.

169.

Bell SL, Bebbington CR, Bushell ME, Sanders PG, Scott MF, Spier RE, Wardell JN (1991) Genetic engineering of cellular physiology. In: Production of Biologicals from Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp304-306. ♦ GS gene used to confer glutamine independence on a hybridoma.

4.

Bell SL, Bushell ME, Scott MF, Wardell JN, Spier RE, Sanders PG (1992) Genetic modification of hybridoma glutamine metabolism: physiological consequences. In: Animal Cell Technology: Developments, Processes and Products. Eds: Spier RE, Griffiths JB, MacDonald C. Butterworth-Heinemann, pp 180-182. ♦ GS gene transfected into hybridoma cell to confer glutamine independence.

5.

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushell ME, Sanders PG (1995) Genetic engineering of hybridoma glutamine metabolism. Enzyme and

65.


Microbial Technol. 17:98-106. ♦ GS gene used to confer glutamine independence on a hybridoma. Bi J -X, Shuttleworth J, Al-Rubeai M (2004) Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells. Biotechnol. Bioeng. 85:741-749.

150.

Browne SM, Al-Rubeai M (2011) Analysis of an artificially selected GS-NS0 variant with increased resistance to apoptosis. Biotechnol. Bioeng. 108:880-892.

201.

Cairo JJ, Paredes C, Godia F, Prats E, Azorin F, Cornudella L (1998) Modification of hybridoma cells metabolism. In: New Developments and New Applications in Animal Cell Technology. Eds: Merten O-W, Perrin P, Griffiths B. Kluwer Academic Publishers, pp167-174.

♦ Transfection of hybridoma with GS gene eliminated glutamine requirement and suppressed ammonia production. Also resulted in halving of glucose uptake rate whilst maintaining similar growth pattern. Discusses metabolic fluxes in cells with and without GS.

86.

Contie M , Leger O, Fouque N, Poitevin Y, Kosco-Vilbois M, Mermod N, Elson G. (2013) IL-17F co-expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering. Biotechnol. Bioeng. 110:1153-1163.

223.

Dorai H. Kyung YS, Ellis D, Kinney C, Lin C, Jan D, Moore G, Betenbaugh MJ (2009) Expression of anti-apoptosis genes alters lactate metabolism of Chinese hamster ovary cells in culture. Biotechnol. Bioeng. 103:592-608.

177.

Dorai H, Ellis D, Keung YS, Campbell M, Zhuang M, Lin C, Betenbaugh MJ (2010) Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines. Biotechnol. Prog. 26:1367-1381.

192.

Fan L , Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye C (2012) Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells. Biotechnol. Bioeng. 109:1007-1015.

207.

Hayes NVL, Smales CM, Klappa P (2010) Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines. Biotechnol. Bioeng. 105:770-779.

186.



111.

Jos sé L , Smales CM, Tuite MF (2010) Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells. Biotechnol. Bioeng. 105:556-566.

184.

Krampe B , Fagan A, Gaora PÓ, Al-Rubeai M (2011) Chemostat-based 199.


transcriptional analysis of growth rate change and BCL-2 over-expression in NS0 cells. Biotechnol. Bioeng. 108:1603-1615. ♦ The GS-NS0 cell line described by Al-Rubeai and co-workers as ‘6A1’ is in fact

the cell line 6A1(100)3 (Bebbington et al 1992, reference #3). O'Connor KC , Muhitch JW, Lacks DJ, Al-Rubeai M (2006) Modelling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells. Biotechnol. Lett. 28:1919-1924. ♦ The GS-NS0 cell line described by these authors is actually the cell line

6A1(100)3 described by Bebbington et al in 1992.

154.

Paredes C, Prats E, Cairo JJ, Azoris F, Cornudella Ll, Godia F (1999) Modification of glucose and glutamine metabolism in hybridoma cells through metabolic engineering. Cytotechnol. 30:85-93. ♦ Transfected a hybridoma cell line with GS. Transfected cells had a reduced

growth rate and a lower glucose utilisation rate. Ammonia and alanine production was eliminated. Alanine was required for optimal growth.

101.

Tey BT , Singh RP, Al-Rubeai M (1999) Influence of bcl-2 over-expression on NS0 and CHO culture viability and chimeric antibody productivity. In: Animal Cell Technology: Products from Cells, Cells as Products. Eds: Bernard A et al. pp 59-61 Kluwer. ♦ Over-expression of bcl-2 significantly reduced the rate of cell death in a GS-

CHO and in GS-NS0 cell line and resulted in a 19% and 25% increase, respectively in antibody production.

98.

Tey BT, Singh RP, Piredda L., Piacentini M, Al-Rubeai M (2000) Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotech. Bioeng. 68:31-43. ♦ Transfection with the control expression vector (Neo marker) used in this study

and exposure to the selective drug G418 led to upregulation of endogenous bcl-2. This led to an increase in cell viability in cell cultures and prolonged survival. There was no influence on antibody titre.

99.

Tey BT , Singh RP, Piredda L, Piacentini MP, Al-Rubeai M (2000) Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures. J. Biotechnol. 79:147-159. ♦ In batch culture, no difference was seen in product concentration between cell

line over-expressing Bcl-2 and control cell line. However, in fed-batch culture using a concentrated amino acid feed, antibody concentration was increased 60% in the Bcl-2 over-expressing cell line..

147.

Tey BT , Al-Rubeai M. (2004) Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity. Apoptosis 9:843-852.

160

Tey BT , Al-Rubeai M (2005) Effect of Bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells. J Biosci Bioeng 100:303-310.

159


Tey BT , Al-Rubeai M. (2005) Bcl-2 over-expression reduced the serum dependency and improved the nutrient metabolism in a NS0 cells culture. Biotechnol. Bioprocess Eng. 10:254-261.

225.

Watanabe S , Shuttleworth J, Al-Rubeai M (2002) Regulation of cell cycle and productivity in NS0 cells by the over-expression of p21CIP1. Biotechnol. Bioeng. 77:1-7.

148.

gs expression system bibliography in progress version...

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